Receptor ligand VEGF-C

ABSTRACT

Provided are polypeptide ligands for the receptor tyrosine kinase, Flt4. Also provided are cDNAs and vectors encoding the ligands, pharmaceutical compositions and diagnostic reagents comprising the ligands, and methods of making and using the ligands.

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/601,132, filed Feb. 14, 1996, which is acontinuation-in-part of U.S. patent application Ser. No. 08/585,895,filed Jan. 12, 1996, now U.S. Pat. No. 6,245,530, which is acontinuation-in-part of U.S. patent application Ser. No. 08/510,133,filed Aug. 1, 1995, now U.S. Pat. No. 6,221,839.

FIELD OF THE INVENTION

The present invention generally relates to the field of geneticengineering and more particularly to growth factors for endothelialcells and growth factor genes.

BACKGROUND OF THE INVENTION

Developmental growth, the remodelling and regeneration of adult tissues,as well as solid tumor growth, can only occur when accompanied by bloodvessel formation. Angioblasts and hematopoietic precursor cellsdifferentiate from the mesoderm and form the blood islands of the yolksac and the primary vascular system of the embryo. The development ofblood vessels from these early (in situ) differentiating endothelialcells is termed vasculogenesis. Major embryonic blood vessels arebelieved to arise via vasculogenesis, whereas the formation of the restof the vascular tree is thought to occur as a result of vascularsprouting from pre-existing vessels, a process called angiogenesis,Risau et al., Devel. Biol., 125:441-450 (1988).

Endothelial cells give rise to several types of functionally andmorphologically distinct vessels. When organs differentiate and begin toperform their specific functions, the phenotypic heterogeneity ofendothelial cells increases. Upon angiogenic stimulation, endothelialcells may re-enter the cell cycle, migrate, withdraw from the cell cycleand subsequently differentiate again to form new vessels that arefunctionally adapted to their tissue environment. Endothelial cellsundergoing angiogenesis degrade the underlying basement membrane andmigrate, forming capillary sprouts that project into the perivascularstroma. Ausprunk et al., Microvasc. Rev., 14:51-65 (1977). Angiogenesisduring tissue development and regeneration depends on the tightlycontrolled processes of endothelial cell proliferation, migration,differentiation, and survival. Dysfunction of the endothelial cellregulatory system is a key feature of many diseases. Most significantly,tumor growth and metastasis have been shown to be angiogenesisdependent. Folkman et al., J. Biol. Chem., 267:10931-10934 (1992).

Key signals regulating cell growth and differentiation are mediated bypolypeptide growth factors and their transmembrane receptors, many ofwhich are tyrosine kinases. Autophosphorylated peptides within thetyrosine kinase insert and carboxyl-terminal sequences of activatedreceptors are commonly recognized by kinase substrates involved insignal transduction for the readjustment of gene expression inresponding cells. Several families of receptor tyrosine kinases havebeen characterized. Van der Geer et al., Ann. Rev. Cell Biol.,10:251-337 (1994). The major growth factors and receptors transducingangiogenic stimuli are schematically shown in FIG. 1.

Fibroblast growth factors are also known to be involved in theregulation of angiogenesis. They have been shown to be mitogenic andchemotactic for cultured endothelial cells. Fibroblast growth factorsalso stimulate the production of proteases, such as collagenases andplasminogen activators, and induce tube formation by endothelial cells.Saksela et al., Ann. Rev. Cell Biol., 4:93-126 (1988). There are twogeneral classes of fibroblast growth factors, FGF-1 and FGF-2, both ofwhich lack conventional signal peptides. Both types have an affinity forheparin and FGF-2 is bound to heparin sulfate proteoglycans in thesubendothelial extracellular matrix from which it may be released afterinjury. Heparin potentiates the stimulation of endothelial cellproliferation by angiogenic FGFs, both by protecting againstdenaturation and degradation and dimerizing the FGFs. Culturedendothelial cells express the FGF-1 receptor but no significant levelsof other high-affinity fibroblast growth factor receptors.

Among other ligands for receptor tyrosine kinases, the platelet derivedgrowth factor, PDGF-BB, has been shown to be weakly angiogenic in thechick chorioallantoic membrane. Risau et al., Growth Factors, 7:261-266(1992). Transforming growth factor α (TGFα) is an angiogenic factorsecreted by several tumor cell types and by macrophages. Hepatocytegrowth factor (HGF), the ligand of the c-met proto-oncogene-encodedreceptor, also is strongly angiogenic.

Recent evidence shows that there are endothelial cell specific growthfactors and receptors that may be primarily responsible for thestimulation of endothelial cell growth, differentiation and certaindifferentiated functions. The best studied of these is vascularendothelial growth factor (VEGF), a member of the PDGF family. Vascularendothelial growth factor is a dimeric glycoprotein of disulfide-linked23 kD subunits. Other reported effects of VEGF include the mobilizationof intracellular calcium, the induction of plasminogen activator andplasminogen activator inhibitor-1 synthesis, stimulation of hexosetransport in endothelial cells, and promotion of monocyte migration invitro. Four VEGF isoforms, encoded by distinct mRNA splice variants,appear to be equally capable of stimulating mitogenesis in endothelialcells. However, each isoform has a different affinity for cell surfaceproteoglycans, which behave as low affinity receptors for VEGF. The 121and 165 amino acid isoforms of VEGF (VEGF121 and VEGF165) are secretedin a soluble form, whereas the isoforms of 189 and 206 amino acidresidues remain cell surface-associated and have a strong affinity forheparin. VEGF was originally purified from several sources on the basisof its mitogenic activity toward endothelial cells, and also by itsability to induce microvascular permeability, hence it is also calledvascular permeability factor (VPF).

The pattern of VEGF expression suggests its involvement in thedevelopment and maintenance of the normal vascular system and in tumorangiogenesis. During murine development, the entire 7.5 day post-coital(p.c.) endoderm expresses VEGF and the ventricular neuroectodermproduces VEGF at the capillary ingrowth stage. See Breier et al.,Development, 114:521-523 (1992). On day two of quail development, thevascularized area of the yolk sac as well as the whole embryo showexpression of VEGF. In addition, epithelial cells next to fenestratedendothelia in adult mice show persistent VEGF expression, suggesting arole in the maintenance of this specific endothelial phenotype andfunction.

Two high affinity receptors for VEGF have been characterized. These areVEGFR-1/Flt-1 (fms-like tyrosine kinase-1) and VEGFR-2/Kdr/Flk-1 (kinaseinsert domain containing receptor/fetal liver kinase-1). Those receptorsare classified in the PDGF-receptor family, but they have seven ratherthan five immunoglobulin-like loops in their extracellular domain andthey possess a longer kinase insert than normally observed in thisfamily. The expression of VEGF receptors occurs mainly in vascularendothelial cells, although some may be present on monocytes andmelanoma cells. Only endothelial cells have been reported to proliferatein response to VEGF, and endothelial cells from different sources showdifferent responses. Thus, the signals mediated through VEGFR-1 andVEGFR-2 appear to be cell type specific. The VEGF-related placentagrowth factor (PlGF) was recently shown to bind to VEGFR-1 with highaffinity. PlGF was able to enhance the growth factor activity of VEGF,but it did not stimulate endothelial cells on its own. Naturallyoccurring VEGF/PlGF heterodimers were nearly as potent mitogens as VEGFhomodimers for endothelial cells.

The Flt4 receptor tyrosine kinase (VEGFR-3) is closely related instructure to the products of the VEGFR-1 and VEGFR-2 genes. Despite thissimilarity, the mature form of Flt4 differs from the VEGF receptors inthat it is proteolytically cleaved in the extracellular domain into twodisulfide-linked polypeptides. Pajusola et al., Cancer Res.,52:5738-5743 (1992). The 4.5 and 5.8 kb Flt4 mRNAs encode polypeptideswhich differ in their C-termini due to the use of alternative 3′ exons.The VEGFs do not show specific binding to Flt4 or induce itsautophosphorylation.

Expression of Flt4 appears to be more restricted than expression ofVEGFR-1 or VEGFR-2. The expression of Flt4 first becomes detectable byin situ hybridization in the angioblasts of head mesenchyme, thecardinal vein, and extraembryonically in the allantois of 8.5 day p.c.mouse embryos. In 12.5 day p.c. embryos the Flt4 signal is observed indeveloping venous and presumptive lymphatic endothelia, but arterialendothelia appear negative. During later stages of development, Flt4mRNA becomes restricted to developing lymphatic vessels. Only thelymphatic endothelia and some high endothelial venules express Flt4 mRNAin adult human tissues and increased expression occurs in lymphaticsinuses in metastatic lymph nodes and in lymphangioma. These resultssupport the theory of the venous origin of lymphatic vessels.

Five endothelial cell specific receptor tyrosine kinases, Flt-1(VEGFR-1), KDR/Flk-1 (VEGFR-2), Flt4, Tie and Tek/Tie-2 have so far beendescribed, which possess the intrinsic tyrosine kinase activityessential for signal transduction. Targeted mutations inactivatingFlt-1, FLk-1, Tie and Tek in mouse embryos have indicated theiressential and specific roles in vasculogenesis and angiogenesis at themolecular level. VEGFR-1 and VEGFR-2 bind VEGF with high affinity (K_(d)16 pM and 760 pM, respectively) and VEGFR-1 also binds the relatedplacenta growth factor (PlGF; K_(d) about 200 pM), while the ligands forTie, Tek, and Flt4 have not heretofore been reported.

SUMMARY OF THE INVENTION

The present invention provides a ligand for the Flt4 receptor tyrosinekinase. Thus, the invention provides a purified and isolated polypeptidewhich is capable of binding to the Flt4 receptor tyrosine kinase.Preferably, an Flt4 ligand of the invention is capable of stimulatingtyrosine phosphorylation of Flt4 receptor tyrosine kinase in a host cellexpressing the Flt4 receptor tyrosine kinase. Preferred ligands of theinvention are mammalian polypeptides. Highly preferred ligands are humanpolypeptides.

In one embodiment, an FlT4 ligand has a molecular weight ofapproximately 23 kD as determined by SDS-PAGE under reducing conditions.For example, the invention includes a ligand composed of one or morepolypeptides of approximately 23 kD is purifyable from conditioned mediafrom a PC-3 prostatic adenocarcinoma cell line, the cell line havingATCC Acc. No. CRL 1435. Amino acid sequencing of this PC-3 cell derivedligand revealed that the ligand comprises an amino terminal amino acidsequence set forth in SEQ ID NO: 13. A conditioned medium comprising anFlt4 ligand is itself an aspect of the invention.

In a highly preferred embodiment, the ligand comprises a fragment of theamino acid sequence shown in SEQ ID NO: 33 which specifically binds tothe human Flt4 receptor tyrosine kinase. Exemplary fragments include: apolypeptide comprising an amino acid sequence set forth in SEQ ID NO: 33from about residue 112 to about residue 213; a polypeptide comprising anamino acid sequence from about residue 104 to about residue 227 of SEQID NO: 33; and a polypeptide comprising an amino acid sequence fromabout residue 112 to about residue 227 of SEQ ID NO: 33. Other exemplaryfragments include polypeptides comprising amino acid sequences of SEQ IDNO: 33 that span, approximately, the following residues: 31-213, 31-227,32-227, 103-217, 103-225, 104-213, 113-213, 103-227, 113-227, 131-211,161-211, 103-225, 227-419, 228-419, 31-419, and 1-419, as described ingreater detail below.

The present invention also provides one or more polypeptide precursorsof an Flt4 ligand, wherein one such precursor (designated“prepro-VEGF-C”) comprises the complete amino acid sequence (amino acidresidues 1 to 419) shown in SEQ ID NO: 33. Thus, the invention includesa purified and isolated polypeptide having the amino acid sequence ofresidues 1 to 419 shown in SEQ ID NO: 33. A putative 102 amino acidleader (prepro) peptide has been identified in the amino acid sequenceshown in SEQ ID NO: 33. Thus, in a related aspect, the inventionincludes a purified and isolated polypeptide having the amino acidssequence of residues 103-419 shown in SEQ ID NO: 33.

In one embodiment, an expressed Flt4 ligand precursor is proteolyticallycleaved upon expression to produce an approximately 23 kD polypeptidewhich is the Flt4 ligand (herein designated VEGF-C). Thus, an Flt4ligand is provided which is the cleavage product of the precursorpeptide shown in SEQ ID NO: 33 and which has a molecular weight ofapproximately 23 kD under reducing conditions.

A putative VEGF-C precursor or 'splice variant, consisting ofpolypeptides with molecular weights of about 29 and 32 kD, also isconsidered an aspect of the invention.

In another embodiment, an expressed Flt4 ligand precursor isproteolytically cleaved upon expression to produce an approximately 21kD VEGF-C polypeptide. Sequence analysis has indicated that an observed21 kD form has an amino terminus approximately 9 amino acids downstreamfrom the amino terminus of the 23 kD form, suggesting alternativecleavage sites exist.

From the foregoing, it will be apparent that an aspect of the inventionincludes a fragment of the purified and isolated polypeptide having theamino acid sequence of residues 1 to 419 shown in SEQ ID NO: 33, thefragment being capable of specifically binding to Flt4 receptor tyrosinekinase. Preferred embodiments include fragments having an apparentmolecular weight of approximately 21/23 kD and 29/32 kD as assessed bySDS-PAGE under reducing conditions.

Evidence suggests that the amino acids essential for retaining Flt4ligand activity are contained within approximately amino acids103/112-226/227 of SEQ ID NO: 33, and that a carboxy-terminalproteolytic cleavage to produce a mature, naturally-occurring Flt4ligand occurs at the approximate position of amino acids 226-227 of SEQID NO: 33. Accordingly, a preferred Flt4 ligand comprises approximatelyamino acids 103-227 of SEQ ID NO: 33.

VEGF-C mutational analysis described herein indicates that a naturallyoccurring VEGF-C polypeptide spanning amino acids 103-227 of SEQ ID NO:33, produced by a natural processing cleavage that defines theC-terminus, exists and is biologically active as an Flt4 ligand. Apolypeptide fragment consisting of residues 104-213 of SEQ ID NO: 33 hasbeen shown to retain VEGF-C biological activity. Additional mutationalanalyses indicate that a polypeptide spanning only amino acids 113-213of SEQ ID NO: 33 retains Flt4 ligand activity. Accordingly, preferredpolypeptides comprise sequences spanning, approximately, amino acidresidues 103-227, 104-213, or 113-213, of SEQ ID NO: 33.

Moreover, sequence comparisons of members of the VEGF family ofpolypeptides provide an indication that still smaller fragments willretain biological activity, and such smaller fragments are intended asaspects of the invention. In particular, eight highly conserved cysteineresidues of the VEGF family of polypeptides define a region fromresidues 131-211 of SEQ ID NO: 33 (see FIGS. 10 & 31B); therefore, apolypeptide spanning from about residue 131 to about residue 211 isexpected to retain VEGF-C biological activity. In fact, a polypeptidecomprising approximately residues 161-211, which retains anevolutionarily-conserved RCXXCC motif, is postulated to retain VEGF-Cactivity, and therefore is intended as an aspect of the invention. Someof the conserved cysteine residues in VEGF-C participate in interchaindisulfide bonding to make homo- and heterodimers of the variousnaturally occurring VEGF-C polypeptides. Beyond the precedingconsiderations, evidence exists that VEGF-C polypeptides lackinginterchain disulfide bonds retain VEGF-C biological activity. Inparticular, VEGF-C that has been reduced and alkylated (processes thatprevent the formation of disulfide bonds by cysteine residues) retainsbiological activity. Consequently, the materials and methods of theinvention include all VEGF-C fragments that retain at least onebiological activity of VEGF-C, regardless of the presence or absence ofinterchain disulfide bonds. The invention also includes multimerscomprising such fragments linked to each other or to other polypeptides.Fragment linkage may be by way of covalent bonding (e.g., disulfidebonding) or non-covalent bonding of polypeptide chains (e.g, hydrogenbonding, bonding due to stable or induced dipole-dipole interactions,bonding due to hydrophobic or hydrophilic interactions, combinations ofthese bonding mechanisms, and the like).

In yet another related aspect, the invention includes variants andanalogs of the aforementioned polypeptides, including VEGF-C, precursorsof VEGF-C, and fragments of VEGF-C. The variants contemplated by theinvention include purified and isolated polypeptides having amino acidsequences that differ from the amino acid sequences of VEGF-C, VEGF-Cprecursors and VEGF-C fragments by conservative substitutions, asrecognized by those of skill in the art, or by additions or deletions ofamino acid residues that are compatible with the retention of at leastone biological activity of VEGF-C.

Analogs contemplated by the invention include polypeptides havingmodifications to one or more amino acid residues that differ from themodifications found in VEGF-C, VEGF-C precursors, or VEGF-C fragments,but are compatible with the retention of at least one biologicalactivity of VEGF-C, VEGF-C precursors, or VEGF-C fragments. For example,analogs within the scope of the invention include glycosylation variantsand conjugants (attachment of the aforementioned polypeptides tocompounds such as labels, toxins, etc.)

The present invention also provides purified and isolatedpolynucleotides (i.e., nucleic acids) encoding novel polypeptides, forexample a cDNA or corresponding genomic DNA encoding VEGF-C. VEGF-C is aligand for the FLT4 receptor tyrosine kinase (VEGFR-3), a receptortyrosine kinase related to VEGFR-1 and VEGFR-2 that does not bind VEGF.VEGFR-3 is expressed in venous and lymphatic endothelia of fetal tissuesand predominantly in lymphatic endothelia of adult tissues. Kaipainenal., Cancer Res., 54:6571-77 (1994); Kaipainen, et al., Proc. Natl.Acad. Sci. (USA), 92:3566-70 (1995). A preferred nucleic acid of theinvention encodes VEGF-C, for example a DNA encoding amino acid residues1 to 419 of SEQ ID NO: 33. Other preferred nucleic acids encode one ofthe aforementioned fragments of VEGF-C. The invention also comprehendsanalogs of these polynucleotides, or derivatives of any one of thesepolynucleotides sufficiently duplicative of the corresponding naturallyoccurring polynucleotide such that the encoded polypeptide retains atleast one biological property of the polypeptide encoded by thenaturally occurring polynucleotide. DNA polynucleotides according to theinvention include genomic DNAs, cDNAs, and oligonucleotides comprisingthe coding sequence for a fragment of VEGF-C, or an analog of a VEGF-Cfragment that retains at least one of the biological activities of aVEGF-C fragment. Distinct polynucleotides encoding a polypeptide of theinvention by virtue of the degeneracy of the genetic code are within thescope of the invention.

A preferred polynucleotide according to the invention comprises thehuman VEGF-C cDNA sequence set forth in SEQ ID NO: 32 from nucleotide352 to 1611. Other polynucleotides according to the invention encode aVEGF-C polypeptide from, e.g., mammals other than humans. Still otherpolynucleotides of the invention comprise a coding sequence for a VEGF-Cfragment, and allelic variants of those DNAs encoding part or all ofVEGF-C. Moreover, the invention comprehends polynucleotides that differfrom native VEGF-C-encoding polynucleotides by the deletion, insertionor substitution of nucleotides and which encode polypeptides that retainpart or all of at least one of the biological activities associated withnative VEGF-C-encoding polynucleotides. Further, the inventioncontemplates polynucleotides having sequences that differ frompolynucleotides encoding a VEGF-C fragment in a manner that results inconservative amino acid differences between the encoded polypeptides, asunderstood by those of skill in the art.

The invention further comprises polynucleotides that hybridize to theabove-defined polynucleotides under standard stringent hybridizationconditions, or which would hybridize but for the degeneracy of thegenetic code. Exemplary stringent hybridization conditions are asfollows: hybridization at 42° C. in 50% formamide, 5×SSC, 20 mM Na.PO₄,pH 6.8 and washing in 0.2×SSC at 55° C. It is understood by those ofskill in the art that variation in these conditions occurs based on thelength and GC nucleotide content of the sequences to be hybridized.Formulas standard in the art are appropriate for determining exacthybridization conditions. See Sambrook et al., Molecular Cloning: ALaboratory Manual (Second ed., Cold Spring Harbor Laboratory Press 1989)§§9.47-9.51. These polynucleotides, capable of hybridizing topolynucleotides encoding VEGF-C, VEGF-C fragments, or VEGF-C analogs,are useful as nucleic acid probes for identifying, purifying andisolating polynucleotides encoding other (non-human) mammalian forms ofVEGF-C. Additionally, these polynucleotides are useful in screeningmethods of the invention, as described below.

Preferred nucleic acid probes of the invention comprise nucleic acidsequences of at least about 16 continuous nucleotides of SEQ ID NO: 32.More preferably, these nucleic acid probes would have at least about 20nucleotides found in a subsequence of SEQ ID NO: 32. In using thesenucleic acids as probes, it is preferred that the nucleic acidsspecifically hybridize to a portion of the sequence set forth in SEQ IDNO: 32. Specific hybridization is herein defined as hybridization understandard stringent hybridization conditions. To identify and isolateother mammalian VEGF-C genes specifically, nucleic acid probespreferably are selected such that they fail to hybridize to genesrelated to VEGF-C (e.g., fail to hybridize to human VEGF or to humanVEGF-B genes).

Thus, the invention comprehends polynucleotides comprising at leastabout 16 nucleotides wherein the polynucleotides are capable ofspecifically hybridizing to a gene encoding VEGF-C, e.g., a human gene.The specificity of hybridization ensures that a polynucleotide of theinvention is able to hybridize to a nucleic acid encoding a VEGF-C underhybridization conditions that do not support hybridization of thepolynucleotide to nucleic acids encoding, e.g., VEGF or VEGF-B. In oneembodiment, polynucleotides of at least about 16 nucleotides, andpreferably at least about 20 nucleotides, are selected as continuousnucleotide sequences found in SEQ ID NO: 32 or the complement of thenucleotide sequence set forth in SEQ ID NO: 32.

Thus, aspects of the invention include purified and isolated nucleicacids encoding polypeptides and polypeptide fragments of the invention;vectors which comprise nucleic acids of the invention; and host cellstransformed or transfected with nucleic acids or vectors of theinvention. For example, in a preferred embodiment, the inventionincludes a purified and isolated nucleic acid (e.g., a DNA or an RNA)encoding an Flt4 ligand precursor. Due to the degeneracy of the geneticcode, numerous such coding sequences are possible, each having in commonthe coding of the amino acid sequence shown in SEQ ID NO: 33. As setforth above, the invention includes polypeptides which comprise aportion of the amino acid sequence shown in SEQ ID NO: 33 and which bindthe Flt4 receptor tyrosine kinase (herein designated VEGFR-3); theinvention also is intended to include nucleic acids encoding thesepolypeptides. Ligand precursors according to the invention, whenexpressed in an appropriate host cell, produce, via cleavage, a peptidewhich binds specifically to the Flt4 receptor tyrosine kinase (VEGFR-3).The nucleotide sequence shown in SEQ ID NO: 32 contains a preferrednucleotide sequence encoding the Flt4 ligand (VEGF-C).

The present invention also provides a cell line which produces an Flt4ligand. In a preferred embodiment, the ligand may be purified andisolated directly from the cell culture medium. Also provided arevectors comprising a DNA encoding the Flt4 ligand, and host cellscomprising the vectors. Preferred vectors of the invention areexpression vectors wherein nucleic acids of the invention areoperatively connected to appropriate promoters and other controlsequences, such that appropriate host cells transformed or transfectedwith the vectors are capable of expressing the Flt4 ligand. A preferredvector of the invention is plasmid pFLT4-L, having ATCC accession no.97231. Such vectors and host cells are useful for recombinantlyproducing VEGF-C polypeptides.

The invention further includes a method of making polypeptides of theinvention. In a preferred method, a nucleic acid or vector of theinvention is expressed in a host cell, and a polypeptide of theinvention is purified from the host cell or the host cell's growthmedium.

In a related embodiment, the invention includes a method of making apolypeptide capable of specifically binding to Flt4 receptor tyrosinekinase, comprising the steps of: (a) transforming or transfecting a hostcell with a nucleic acid of the invention; (b) cultivating the host cellto express the nucleic acid; and (c) purifying a polypeptide capable ofspecifically binding to Flt4 receptor tyrosine kinase from the host cellor from the host cell's growth media.

The invention also is intended to include purified and isolatedpolypeptide ligands of Flt4 produced by methods of the invention.

In another aspect, the invention includes an antibody which isspecifically reactive with polypeptides of the invention, such as anFlt4 receptor tyrosine kinase ligand. Antibodies, both monoclonal andpolyclonal, may be made against a ligand of the invention according tostandard techniques in the art. Such antibodies may be used indiagnostic applications to monitor angiogenesis, vascularization,lymphatic vessels and their disease states, wound healing, or certainhematopoietic or leukemia cells, or they may be used to block oractivate the Flt4 receptor.

Ligands according to the invention may be labeled with a detectablelabel and used to identify their corresponding receptors in situ.Labeled Flt4 ligand and anti-Flt4 ligand antibodies may be used asimaging agents in the detection of lymphatic vessels, high endothelialvenules, and Flt4 receptors expressed in histochemical tissue sections.The ligand or antibody may be covalently or non-covalently coupled to asuitable supermagnetic, paramagnetic, electron dense, echogenic, orradioactive agent for imaging. Other, non-radioactive labels, such asbiotin and avidin, may also be used.

A related aspect of the invention is a method for the detection ofspecific cells, e.g., endothelial cells. These cells may be found invivo, or in ex vivo biological tissue samples. The method of detectioncomprises the steps of exposing a biological tissue comprising, e.g.,endothelial cells, to a polypeptide according to claim 1, underconditions wherein the polypeptide binds to the cells, optionallywashing the biological tissue, and detecting the polypeptide bound tothe cells in the biological tissue, thereby detecting the cells.

The present invention also provides diagnostic and clinical applicationsfor claimed ligands. In a preferred embodiment, Flt4 ligands orprecursors are used to accelerate angiogenesis, e.g., during woundhealing, or to promote the endothelial functions of lymphatic vessels. Autility for VEGF-C is suggested as an inducer of angiogenesis also intissue transplantation, in eye diseases, in the formation of collateralvessels around arterial stenoses and into injured tissues afterinfarction. Ligands may be applied in any suitable manner using anappropriate pharmaceutically-acceptable vehicle, e.g., apharmaceutically-acceptable diluent, adjuvant, excipient or carrier.Ligands also may be used to quantify future metastatic risk by assayingbiopsy material for the presence of active receptors or ligands in abinding assay or kit using detectably-labeled ligand. An Flt4 ligandaccording to the invention also may be used to promote re-growth orpermeability of lymphatic vessels in, for example, organ transplantpatients. In addition, an Flt4 ligand may be used to mitigate the lossof axillary lymphatic vessels following surgical interventions in thetreatment of cancer (e.g., breast cancer). Ligands according to theinvention also may be used to treat or prevent inflammation, edema,aplasia of the lymphatic vessels, lymphatic obstruction, elephantiasis,and Milroy's disease. Finally, Flt4 ligands may be used to stimulatelymphocyte production and maturation, and to promote or inhibittrafficking of leukocytes between tissues and lymphatic vessels or toaffect migration in and out of the thymus.

An embodiment of this aspect of the invention is a method of screeningfor an endothelial cell disorder in a mammalian subject. The methodcomprises providing a sample of endothelial cells from the subject,contacting the sample of endothelial cells with a polypeptide accordingto claim 4, determining the growth rate of the cells, and correlatingthe growth rate with a disorder. In a preferred embodiment, theendothelial cells are lymphatic cells. In another preferred embodiment,the mammalian subject is a human being and the endothelial cells arehuman cells. In yet another preferred embodiment, the disorder is avessel disorder, e.g., a lymphatic vessel disorder, such as the loss oflymphatic vessels through surgery or the reduction in function ofexisting lymphatic vessels due to blockages. In another embodiment, theendothelial cells are contacted with the polypeptide in vitro. Thegrowth rate determined in the method is the rate of cell division perunit time, determined by any one of a number of techniques known in theart. The correlation of the growth rate with a disorder can involve apositive or negative correlation, e.g., whether the polypeptide has Flt4ligand activity or is an inhibitor of such activity, as described below.

Inhibitors of the Flt4 ligand may be used to control endothelial cellproliferation and lymphangiomas. For example, such inhibitors may beused to arrest metastatic growth or spread, or to control other aspectsof endothelial cell expression and growth. Inhibitors includeantibodies, antisense oligonucleotides, and peptides which block theFlt4 receptor, all of which are intended as aspects of the invention.

In another embodiment, the invention provides a method for modulatingthe growth of endothelial cells in a mammalian subject comprising thesteps of exposing mammalian endothelial cells to a polypeptide accordingto the invention in an amount effective to modulate the growth of themammalian endothelial cells. In one embodiment, the modulation of growthis effected by using a polypeptide capable of stimulating tyrosinephosphorylation of Flt4 receptor tyrosine kinase in a host cellexpressing the Flt4 receptor tyrosine kinase. In modulating the growthof endothelial cells, the invention contemplates the modulation ofendothelial cell-related disorders. Endothelial cell disorderscontemplated by the invention include, but are not limited to, physicalloss of lymphatic vessels (e.g., surgical removal of axillary lymphtissue), lymphatic vessel occlusion (e.g., elephantiasis), andlymphangiomas. In a preferred embodiment, the subject, and endothelialcells, are human. The endothelial cells may be provided in vitro, or invivo. An effective amount of a polypeptide is defined herein as thatamount of polypeptide empirically determined to be necessary to achievea reproducible change in cell growth rate (as determined by microscopicor macroscopic visualization and estimation of cell doubling time, ornucleic acid synthesis assays), as would be understood by one ofordinary skill in the art.

The present invention also provides methods for using the claimednucleic acids (i.e., polynucleotides) in screening for endothelial celldisorders. In a preferred embodiment, the invention provides a methodfor screening an endothelial cell disorder in a mammalian subjectcomprising the steps of providing a sample of endothelial cell nucleicacids from the subject, contacting the sample of endothelial cellnucleic acids with a polynucleotide according to claim 35, determiningthe level of hybridization between the endothelial cell nucleic acidsand the polynucleotide, and correlating the level of hybridization witha disorder. A preferred mammalian subject, and source of endothelialcell nucleic acids, is a human. The disorders contemplated by the methodof screening with polynucleotides include, but are not limited to,vessel disorders such as the aforementioned lymphatic vessel disorders,and hypoxia.

Purified and isolated polynucleotides encoding other (non-human)mammalian VEGF-C forms also are aspects of the invention, as are thepolypeptides encoded thereby, and antibodies that are specificallyimmunoreactive with the non-human VEGF-C variants. Thus, the inventionincludes a purified and isolated mammalian VEGF-C polypeptide, and alsoa purified and isolated polynucleotide encoding such a polypeptide.

In one embodiment, the invention includes a purified and isolatedpolypeptide having the amino acid sequence of residues 1 to 415 of SEQID NO: 41, which sequence corresponds to a putative mouse VEGF-Cprecursor. The putative mouse VEGF-C precursor is believed to beprocessed into a mature mouse VEGF-C in a manner analogous to theprocessing of the human prepro-polypeptide. Thus, in a related aspect,the invention includes a purified and isolated polypeptide capable ofspecifically binding to an Flt4 receptor tyrosine kinase (e.g., a humanor mouse Flt-4 receptor tyrosine kinase), the polypeptide comprising afragment of the purified and isolated polypeptide having the amino acidsequence of residues 1 to 415 of SEQ ID NO: 41, the fragment beingcapable of specifically binding to the Flt4 receptor tyrosine kinase.The invention further includes purified and isolated nucleic acidsencoding the foregoing polypeptides, such as a nucleic acid comprisingall or a portion of the sequence shown in SEQ ID NO: 40.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic diagram showing major endothelial cell receptortyrosine kinases and growth factors involved in vasculogenesis andangiogenesis.

FIGS. 2A and 2B schematically depict the construction of the pLTRFlt4lexpression vector.

FIG. 3 schematically depicts the construction of the baculovirus vectorencoding a secreted soluble Flt4 extracellular domain (Flt4EC).

FIG. 4 shows results of stimulation of Flt4 autophosphorylation byconditioned medium from PC-3 cell cultures.

FIGS. 5A, 5B, and 5C show that the major tyrosyl phosphorylatedpolypeptide of Flt4-transfected cells stimulated with PC-3 conditionedmedium is the 125 kD Flt4 polypeptide (VEGFR-3), and also that the Flt4stimulating activity is not adsorbed to heparin-sepharose.

FIG. 6 shows Western immunoblotting analysis of the Flt4 Ligand activityisolated from PC-3 conditioned medium.

FIG. 7 shows results of gel electrophoresis of chromatographic fractionsfrom the affinity purification of Flt4 ligand (VEGF-C) isolated fromPC-3 conditioned medium.

FIG. 8 shows results of Western analysis of Flt4 autophosphorylationinduced by either the Flt4 ligand (VEGF-C), VEGF, or PlGF.

FIG. 9 schematically depicts the cloning and analysis of the Flt4ligand, VEGF-C. The VEGF homologous region (dark shaded box) and aminoand carboxyl terminal propeptides (light shaded and unshaded boxes,respectively) as well as putative signal sequence (ss) are depictedbetween 5′ and 3′ untranslated (ut) nucleic acid regions. The cleavagesites for the signal sequence and the amino and carboxyl terminalpropeptides are indicated with triangles.

FIGS. 10A-10C depict a comparison of the deduced amino acid sequences ofPDGEL (SEQ ID NO:52), PDGF-B (SEQ ID NO:53), PlGF-1 (SEQ ID NO:54),VECF₁₆₅ (SEQ ID NO: 55), VECF₁₆₇ (SEQ ID NO:56), and Flt4 ligand (VEGFC(SEQ ID NO: 33)

FIG. 11 shows the stimulation of autophosphorylation of the Flt4receptor by conditioned medium from cells transfected with the pREP7expression vector containing the VEGF-C-encoding cDNA insert of plasmidFLT4-L.

FIG. 12 shows Northern blotting analysis of the genes encoding VEGF,VEGF-B, AND VEGF-C (indicated by “FLT4-L”) in two human tumor celllines.

FIG. 13A is an autoradiograph showing recombinant VEGF-C isolatedfollowing a pulse-chase experiment and electrophoresed via SDS-PAGEunder reducing conditions.

FIG. 13B is a photograph of polyacrylamide gel showing that recombinantVEGF-C forms are disulfide-linked in nonreducing conditions.

FIGS. 14A and 14B depict Western blots showing that VEGF-C stimulatesautophosphorylation of VEGFR-2 (KDR) but has no effect on PDGFR-βphosphorylation.

FIGS. 15A, 15B, and 15C show that VEGF-C stimulates endothelial cellmigration in a three-dimensional collagen gel assay.

FIG. 16A shows the expression of VEGF-C mRNA in human adult tissues.

FIG. 16B shows the expression of VEGF, VEGF-B, and VEGF-C in selectedhuman fetal tissues.

FIG. 17 schematically depicts the chromosomal localization of the VEGF-Cgene.

FIG. 18 is a Northern blot hybridization study showing the effects ofhypoxia on the mRNA expression of VEGF (VEGF-A), VEGF-B and VEGF-C.

FIG. 19 depicts autoradiograms from a pulse-chase immunoprecipitationexperiment wherein cells transfected with a VEGF-C expression vector(VEGF-C) and mock transfected cells (M) were pulse-labeled withradioactive amino acids and chased for varying lengths of time.

FIG. 20 is a schematic map of the K14-VEGF-C vector construct. Thejunction sequences depicted therein are set forth in Seq. ID. NO:51 and52.

FIGS. 21A-C depict electrophoretic fractionations of the various formsof recombinant VEGF-C produced by transfected 293 EBNA cells. FIG. 21Bdepicts the electrophoretic fractionation, under non-reducingconditions, of polypeptides produced from mock (M) transfected cells,cells transformed with wild type (wt) VEGF-C cDNA, and cells transfectedwith a cDNA variant encoding VEGF-C-R102S. Each of the bands identifiedin FIG. 21B was excised and electrophoretically fractionated in aseparate lane under reducing conditions. Fractionation of bandscorresponding to wt VEGF-C are depicted in FIG. 21A; fractionation ofbands corresponding to the R102S variant are depicted in FIG. 21C.

FIGS. 22A-B depict the forms and sizes of wild type and mutantrecombinant VEGF-Cs, as revealed by non-reducing gel electrophoresis.FIG. 22A shows the VEGF-C forms secreted into the media; FIG. 22B showsthe VEGF-C forms retained by the cells. Mock (M) transfected cellsserved as a control.

FIGS. 23A-B present a comparison of the pattern of immunoprecipitated,labelled VEGF-C forms using antisera 882 and antisera 905. Adjacentlanes contain immunoprecipitates that were (lanes marked +) or were not(lanes marked −) subjected to reduction and alkylation.

FIGS. 24A-B present Northern blots of total RNA isolated from cellsgrown in the presence or absence of interleukin-1 (IL-1) and/ordexamethasone (DEX) for the indicated times. For FIG. 25B, the Northernblot was probed with radiolabeled DNA from a VEGF 581 bp cDNA coveringbps 57-638 (Genbank Acc. No. X15997), and a human VEGF-B₁₆₇ cDNAfragment (nucleotides 1/382, Genbank Acc. No. U48800). For FIG. 24A, theNorthern blot was probed with radiolabeled DNA from a human full-lengthVEGF-C cDNA (Genbank Acc. No. X94216). 18S and 28S-rRNA markers.

FIG. 25 illustrates the cloning strategy for inserting a DNA encoding atruncated VEGF-C containing amino acid residues 104-213 SEQ ID NO: 33into the Pichia pastoris expression vector pHIL-S1.

FIGS. 26A-B show VEGF-C expression in P. pastoris cultures transfectedwith a VEGF-C cDNA, with vector alone, or mock-(M) transfected,following induction with methanol for various periods of time asindicated. About 10 μl of medium was analyzed by gel electrophoresisfollowed by Western blotting and detection with anti-VEGF-C antiserum.FIG. 26B depicts the results of a Western blot wherein NIH 3T3 cellsexpressing VEGFR-3 (Flt4), and PAE cells expressing VEGFR-2 (KDR), werestimulated with 5× concentrated medium from Pichia yeast transfectedwith a VEGF-C cDNA-containing vector (+), with a vector lacking aninsert (−), or stimulated with the positive control vanadate. Thestimulated cells were lysed and immunoprecipitated with VEGFR-specificantibodies, and the immunoprecipitates were blotted and probed withanti-phosphotyrosine antibodies.

FIGS. 27A-B present gel electrophoretograms of human VEGF-C (wt) andVEGF-C variants secreted (FIG. 27A) or retained (FIG. 27B) by the host293 EBNA cells. Mock (M) transfected cells served as a control.Molecular weight markers are indicated on the left in kilodaltons (kD).

FIGS. 28A-B show Western blots of VEGFRs that were stimulated toautophosphorylate by wild type (wt) VEGF-C, as well as three VEGF-Cpolypeptide variants. Cell lysates (NIH 3T3 for VEGFR-3 and PAE forVEGFR-2) were subjected to receptor-specific antisera and the receptorswere immunoprecipitated. Immunoprecipitates were then gel-fractionatedand blotted for Western analyses. Western blots were probed withanti-phosphotyrosine antibodies.

FIGS. 29A-D are photomicrographs of hematoxylin-eosin stained sectionsof K14-VEGF-C transgenic and control mouse littermate tissues. Areasshown are from the dorsal skin and snout, as indicated. The white arrowsshow the endothelium-lined margin of the lacunae devoid of red cells.

FIG. 30 presents a Northern blot of polyadenylated RNA from theindicated tissues, hybridized with a pool of VEGF, VEGF-B₁₆₇, and VEGF-Cprobes. Estimated transcript sizes are shown on the right in kilobases(kb).

FIG. 31A provides a schematic illustration of the structure of mouseVEGF-C cDNA clones. The human VEGF-C cDNA structure is shown on the topline, with the signal sequence (SS), N-terminal propeptide (N), VEGF-and Balbiani Ring 3. protein (BR3P) homologous regions indicated. Thelengths of the 5′ and 3′ noncoding regions and the long open readingframe are given in base pairs. The “ATG” and “TAA” in FIG. 31A indicatethe translational start and stop codons, respectively; AAA is thepolyadenylation sequence; and Δ is the site of a 12 bp deletion in themouse cDNA, relative to the human cDNA.

FIG. 31B presents a comparison of the human SEQ ID NO: 33 and mouse SEQID NO: 41 VEGF-C amino acid sequences. The amino acid sequence of mouseVEGF-C is presented on the top line and differences in the humansequence are marked below it. The sequences have been labeled to depictthe regions shown in FIG. 32A. The arrow indicates the putative cleavagesite for the signal peptidase; BR3P motifs, as well as a CR/SC motif,are boxed; and conserved cysteine residues are marked in bold above thesequence. Arginine residue 158 is also marked in bold. The numberingrefers to mouse VEGF-C residues.

FIG. 32A presents SDS-PAGE-fractionated samples immunoprecipitated oraffinity-purified from various ³⁵S-labeled media. In the left panel,control medium from Bosc23 cells containing vector only, medium fromcells expressing human VEGF-C, and medium from cells expressing mouseVEGF-C were independently precipitated with human VEGFR-3-ExtracellularDomain coupled to sepharose. In the right panel, similar conditionedmedia were subjected to precipitation with anti-VEGF-C antibodies. mwm:molecular weight markers; m—mouse; h—human; α—anti.

FIGS. 32B and 32C shows Western blots of gel-fractionatedimmunoprecipitates from lysates made from NIH 3T3 cells expressingVEGFR-3 that had been stimulated by contact with VEGF-C-containinglysates (or a vector control), as a measure of VEGF-C-induced receptorautophosphorylation. Western blots were probed with anti-phosphotyrosine(α-PTyr) or anti-receptor antisera (anti-VEGFR-3 and anti-VEGFR-2), asindicated. As a control, receptor autophosphorylation was induced bypervanadate treatment (VO4). The arrows and numbers refer to theapparent molecular weights of the tyrosyl phosphorylated receptorpolypeptide bands. bVEGF: human baculoviral VEGF-C protein; C-FGEVm:lysate from cells harboring a mouse VEGF-C cDNA cloned into the vectorin an antisense orientation.

FIG. 33A depicts Northern blots of mRNA isolated from the indicatedtissues of adult mice, probed with a VEGF-C probe (top panel), a VEGFR-3probe (middle panel), and with a pool of the VEGF and VEGF-B probes(lower panel). The sizes of the RNA bands are indicated in kilobases(kb) to the right.

FIG. 33B depicts a Northern blot of mouse embryonic mRNA isolated at theindicated gestational ages and probed with a VEGF-C probe.

FIGS. 34A-D depict photomicrographs of in situ hybridizations revealingthe expression of VEGF-C and VEGF-B mRNAs in a parasagittal section of a12.5 day mouse embryo. FIG. 34A: VEGF-C probe; j—jugular veins,mn—metanephros, m—mesenterium (arrowheads), vc—intervertebral vessels,lu—lung (arrowheads). FIG. 34B: VEGF-B probe; h—heart, nasopharyngealarea (arrowheads). FIG. 34C: VEGF-C sense strand probe serving as acontrol. FIG. 34D: bright-field photomicrograph of the same field shownin FIG. 34C.

FIGS. 35A-H depict sections of mouse embryos providing comparisons ofVEGF-C and VEGFR-3 expression in the jugular vessels and the mesentericarea. FIGS. 35A and 35C show expression of VEGF-C transcripts in themesenchyme around the large sac-like structures in the jugular area(arrowheads). FIGS. 35B and 35D show expression of VEGFR-3 transcriptsin the jugular venous sacs. FIGS. 35E and 35G show VEGF-C mRNAdistribution in the mesenteric region of a 14.5 day p.c. embryo, as wellas around the gut. FIGS. 35F and 35H show VEGFR-3 mRNA in the mesentericregion of a 14.5 day embryo, as well as the gut area, developinglymphatic vessels, and venules.

FIGS. 36A-D depict photomicrographs showing FLT4 and VEGF-C in situhybridization of the cephalic region of a 16-day p.c. mouse embryo. Asection of the cephalic region hybridized with the Flt4 probe (FIG. 36A)shows the developing snout, nasal structures and eyes. A more caudallylocated section shows hybridization with the VEGF-C probe (FIG. 36B).The round structures on both sides in the upper part represent thedeveloping molars. In the upper (dorsal) part on both sides of themidline, the caudal portion of the developing conchae are seen. Thesestructures also are shown in higher magnification darkfield (FIG. 36C)and lightfield (FIG. 36D) microscopy.

FIG. 37 presents a schematic illustration of VEGF-C processing,including the major forms of VEGF-C.

DETAILED DESCRIPTION OF THE INVENTION

Described herein is the isolation of a novel vascular endothelial growthfactor and the cloning of a DNA encoding this novel growth factor from acDNA library prepared from the human prostatic adenocarcinoma cell linePC-3. The isolated cDNA encodes a protein which is proteolyticallyprocessed and secreted to cell culture medium. The secreted protein,designated VEGF-C, binds to the extracellular domain of Flt4 and inducestyrosine autophosphorylation of Flt4 and VEGFR-2. VEGF-C also stimulatesthe migration of endothelial cells in collagen gel.

The present invention also is directed to novel growth factorpolypeptides which are ligands for the Flt4 receptor tyrosine kinase(VEGFR-3). Ligands of the invention are members of a family ofplatelet-derived growth factors/vascular endothelial growth factorswhich promote mitosis and proliferation of vascular endothelial cellsand/or mesodermal cells. As described in greater detail in Examples 4and 5, ligands recognizing the Flt4 receptor tyrosine kinase werepurified from a PC-3 prostatic adenocarcinoma cell line (ATCC CRL1435).When applied to a population of cells expressing the Flt4 receptor,ligands of the invention stimulate autophosphorylation, resulting inreceptor activation.

A ligand according to the invention may be expressed as a largerprecursor which is cleaved to produce the ligand. A coexpressed regionin some cases results from alternative splicing of RNA of the ligandgene. Such a co-expressed region may be a function of the particularexpression system used to obtain the ligand. The skilled artisanunderstands that in recombinant production of proteins, additionalsequence may be expressed along with a functional peptide depending uponthe particular recombinant construct used to express the protein, andsubsequently removed to obtain the desired ligand. In some cases therecombinant ligand can be made lacking certain residues of theendogenous/natural ligand. Moreover, it is well-known in thatconservative replacements may be made in a protein which do not alterthe function of the protein. Accordingly, it is anticipated that suchalterations are within the scope of the claims. Moreover, it isanticipated that one or more VEGF-C precursors (the largest putativenative secreted VEGF-C precursor having the complete amino acid sequencefrom residue 32 to residue 419 of SEQ ID NO: 33) is capable ofstimulating the Flt4 ligand without any further processing, in a mannersimilar to that in which VEGF stimulates its receptor in its unprocessedform after the secretion and concomitant release of the signal sequence.

Results reported herein show that Flt4 (VEGFR-3) transmits signals forthe VEGF-C novel growth factor. This conclusion is based on the specificbinding of VEGF-C to recombinant Flt4EC (Flt4 extracellular domain)protein and the induction of VEGFR-3 autophosphorylation by medium fromVEGF-C transfected cells. In contrast, neither VEGF nor PLGF showedspecific binding to VEGFR-3 or induced its autophosphorylation.

As set forth in greater detail below, the putative prepro-VEGF-C has adeduced molecular mass of 46,883; a putative prepro-VEGF-C processingintermediate has an observed molecular weight of about 32 kD; and matureVEGF-C isolated form conditioned media has a molecular weight of about23 kD as assessed by SDS-Page under reducing conditions. A major part ofthe difference in the observed molecular mass of the purified andrecombinant VEGF-C and the deduced molecular mass of the prepro-VEGF-Cencoded by the VEGF-C open reading frame (ORF) is attributable toproteolytic removal of sequences at the amino-terminal andcarboxyl-terminal regions of the prepro-VEGF-C polypeptide. However,proteolytic cleavage of the putative 102 amino acid leader sequence isnot believed to account for the entire difference between the deducedmolecular mass of 46,883 and the observed mass of about 23 kD, becausethe deduced molecular weight of a polypeptide consisting of amino acids1-317 of SEQ ID NO: 33 is 35,724 kD. It is believed that a portion ofthe observed difference in molecular weights is attributable toproteolytic removal of amino acid residues in the amino and carboxylterminal regions of the VEGF-C precursor. By extrapolation from studiesof the structure of PDGF (Heldin et al., Growth Factors, 8:245-52(1993)), it may be that the region critical for receptor binding andactivation by VEGF-C is contained within amino acids residues 104-213,which are found in the secreted form of the VEGF-C protein (i.e., theform lacking the putative prepro leader sequence and somecarboxyterminal sequences). The 23 kD polypeptide binding VEGFR-3 islikely to represent the VEGF-homologous domain. After biosynthesis, thenascent VEGF-C polypeptide may be glycosylated at three putativeN-linked glycosylation sites identified in the deduced VEGF-C amino acidsequence. Polypeptides containing modifications, such as N-linkedglycosylations, are intended as aspects of the invention.

The carboxyl terminal amino acid sequences, which increase the length ofthe VEGF-C polypeptide in comparison with other ligands of this family,show a pattern of spacing of cysteine residues reminiscent of theBalbiani ring 3 protein (BR3P) sequence (Dignam et al., Gene, 88:133-40(1990); Paulsson et al., J. Mol. Biol., 211:331-49 (1990)). This novelC-terminal silk protein-like structural motif of VEGF-C may fold into anindependent domain, which, on the basis of the considerations above, isat least partially cleaved off after biosynthesis. Interestingly, atleast one cysteine motif of the BR3P type is also found in the carboxylterminus of VEGF. In our experiments both the putative precursor andcleaved ligand were detected in the cell culture media, althoughprocessing was apparently cell-associated on the basis of thepulse-chase experiments. The determination of the amino-terminal andcarboxy-terminal sequences of VEGF-C isolates allows the identificationof the proteolytic processing sites. The generation of antibodiesagainst different parts of the pro-VEGF-C molecule will allow the exactdetermination of the precursor-product relationship and ratio, theircellular distribution, and the kinetics of processing and secretion.

VEGF-C has a conserved pattern of eight cysteine residues, which mayparticipate in the formation of intra- and interchain disulfide bonds,creating an antiparallel dimeric biologically active molecule, similarto PDGF. Mutational analysis of the cysteine residues involved in theinterchain disulfide bridges has shown that, in contrast to PDGF, VEGFdimers need to be held together by these covalent interactions in orderto maintain biological activity. Disulfide linking of the VEGF-Cpolypeptide chains was evident in the analysis of VEGF-C in nonreducingconditions.

VEGFR-3, which distinguishes between VEGF and VEGF-C, is closely relatedin structure to VEGFR-1 and VEGFR-2. Finnerty et al., Oncogene,8:2293-98 (1993); Galland et al., Oncogene, 8:1233-40 (1993); Pajusolaet al., Cancer Res., 52:5738-43 (1992). However, the mature form ofVEGFR-3 differs from the two other VEGFRs in that it is proteolyticallycleaved in the extracellular domain into two disulfide-linkedpolypeptides. Pajusola et al., Oncogene, 9:3545-55 (1994). Anotherdifference is that 4.5 and 5.8 kb VEGFR-3 mRNAs encode polypeptidesdiffering in their C-termini and apparently in their signallingproperties due to the use of alternative 3′ exons. Borg et al.,Oncogene, 10:973-84 (1995); Pajusola et al., Oncogene, 8:2931-37 (1993).

Besides VEGFR-3, VEGFR-2 tyrosine kinase also is activated in responseto VEGF-C. VEGFR-2 mediated signals cause striking changes in themorphology, actin reorganization and membrane ruffling of porcine aorticendothelial cells overexpressing this receptor. In these cells, VEGFR-2also mediated ligand-induced chemotaxis and mitogenicity. Waltenbergeret al., J. Biol. Chem., 269:26988-95 (1994). Similarly, the receptorchimera CSF-1R/VEGFR-3 was mitogenic when ectopically expressed in NIH3T3 fibroblastic cells, but not in porcine aortic endothelial cells(Pajusola et al., 1994). Consistent with such results, the bovinecapillary endothelial (BCE) cells, which express VEGFR-2 mRNA but verylittle or no VEGFR-1 or VEGFR-3 mRNAs, showed enhanced migration whenstimulated with VEGF-C. Light microscopy of the BCE cell cultures incollagen gel also suggested that VEGF-C stimulated the proliferation ofthese cells. The data thus indicate that the VEGF ligands and receptorsshow a great specificity in their signalling, which may becell-type-dependent.

The expression pattern of the VEGFR-3 (Kaipainen et al., Proc. Natl.Acad. Sci. (USA), 92:3566-70 (1995)) suggests that VEGF-C may functionin the formation of the venous and lymphatic vascular systems duringembryogenesis. Constitutive expression of VEGF-C in adult tissues shownherein further suggests that this gene product also is involved in themaintenance of the differentiated functions of the lymphatic endotheliumwhere VEGFR-3 is expressed (Kaipainen et al., 1995). Lymphaticcapillaries do not have well-formed basal laminae and an interestingpossibility remains that the silk-like BR3P motif is involved inproducing a supramolectilar structure which could regulate theavailability of VEGF-C in tissues. However, as shown here, VEGF-C alsoactivates VEGFR-2, which is abundant in proliferating endothelial cellsof vascular sprouts and branching vessels of embryonic tissues, but notso abundant in adult tissues. Millauer et al., Nature, 367:576-78(1993). These data have suggested that VEGFR-2 is a major regulator ofvasculogenesis and angiogenesis. VEGF-C may thus have a unique effect onlymphatic endothelium and a more redundant function, shared with VEGF,in angiogenesis and possibly in regulating the permeability of severaltypes of endothelia. Because VEGF-C stimulates VEGFR-2 and promotesendothelial migration, VEGF-C may be useful as an inducer ofangiogenesis of blood and lymphatic vessels in wound healing, in tissuetransplantation, in eye diseases, and in the formation of collateralvessels around arterial stenoses and into injured tissues afterinfarction.

Taken together, these results show an increased complexity of signallingin the vascular endothelium. They reinforce the concept that when organsdifferentiate and begin to perform their specific functions, thephenotypic heterogeneity of endothelial cells increases in several typesof functionally and morphologically distinct vessels. However, uponstimulation by suitable angiogenic stimuli, endothelial cells canre-enter the cell cycle, migrate, withdraw from the cell cycle andsubsequently differentiate again to form new vessels that arefunctionally adapted to their tissue environment. This process ofangiogenesis, concurrent with tissue development and regeneration,depends on the tightly controlled balance between positive and negativesignals for endothelial cell proliferation, migration, differentiationand survival.

Previously-identified growth factors promoting angiogenesis include thefibroblast growth factors, hepatocyte growth factor/scatter factor, PDGFand TGF-α. (See e.g., Folkman, Nature Med., 1:27-31 (1995); Friesel etal., FASEB J., 9:919-25 (1995); Mustonen et al., J. Cell. Biol.,129:895-98 (1995). However, VEGF has been the only growth factorrelatively specific for endothelial cells. The newly identified factorsVEGF-B and VEGF-C thus increase our understanding of the complexity ofthe specific and redundant positive signals for endothelial cellsinvolved in vasculogenesis, angiogenesis, permeability, and perhaps alsoother endothelial functions.

Also described herein is the localization of the VEGF-C gene in humanchromosomes by analysis of somatic cell hybrids and fluorescence in situhybridization (FISH). Southern blotting and polymerase chain reactionanalysis of somatic cell hybrids and fluorescence in situ hybridizationof metaphase chromosomes were used to assess the chromosomallocalization of the VEGF-C gene. The VEGF-C gene was located onchromosome 4q34, close to the human aspartylglucosaminidase genepreviously mapped to 4q34-35. The VEGF-C locus at 4q34 is a candidatetarget for mutations leading to vascular malformations or cardiovasculardiseases. Expression studies using Northern blotting show abundantVEGF-C expression in heart and skeletal muscle; other tissues, such asplacenta ovary, small intestine, thyroid gland, kidney, prostate,spleen, testis and large intestine also express this gene. Whereas PlGFis predominantly expressed in the placenta, the expression patterns ofVEGF, VEGF-B and VEGF-C overlap in many tissues, which suggests thatthey may form heterodimers and interact to exert their physiologicalfunctions.

Targeted mutagenesis leading to inactivation of the VEGF receptor lociin the mouse genome has shown that VEGFR-1 is necessary for the properorganization of endothelial cells forming the vascular endothelium,while VEGFR-2 is necessary for the generation of both endothelial andhematopoietic cells. This suggests that the four genes of the VEGFfamily can be targets for mutations leading to vascular malformations orcardiovascular diseases.

The following Examples illustrate preferred embodiments of theinvention, wherein the isolation, characterization, and function of Flt4ligands and ligand-encoding nucleic acids according to the invention areshown.

EXAMPLE 1 Production of pLTRFlt4l Expression Vector

Construction of the LTR-Flt4l vector is schematically shown in FIGS. 2Aand 2B. The full-length Flt4s cDNA (Genbank Accession No. X68203, SEQ IDNO: 36) was assembled by first subcloning the S2.5 fragment, reported inPajusola et al., Cancer Res., 52:5738-5743 (1992), incorporated byreference herein, containing base pairs 56-2534 of the Flt4s into theEcoRI site of the pSP73 vector (Promega, Madison, Wis.).

Since cDNA libraries used for screening of Flt4 cDNAs did not containthe extreme 5′ protein-coding sequences, inverse PCR was used for theanplification of the 5′ end of Flt4 corresponding to the first 12 aminoacid residues (MQRGAALCLRLW). Poly(A)⁺ RNA was isolated from human HELerythroleukemia cells and double-stranded cDNA, were synthesized usingan Amersham cDNA Synthesis System Plus kit (Amersham Corp.,Buckinghamshire, U.K.) and a gene-specific primer:5′-TGTCCTCGCTGTCCTTGTCT-3′ (SEQ ID NO: 1), which was located 195 bpdownstream of the 5′ end of clone S2.5. Double-stranded cDNA was treatedwith T4 DNA polymerase to blunt the ends and cDNA was purified byfiltration with Centricon 100 filters (Amicon Inc., Beverly, Mass.).Circularization of the blunt-ended cDNA was performed by ligation in atotal volume of 150 microliters. The reaction mixture contained astandard ligation buffer, 5% PEG-8000, 1 mM DTT and 8 U of T4 DNA ligase(New England Biolabs, Beverly, Mass.). Ligation was carried out at 16°C. for 16 hours. Fifteen microliters of this reaction mix were used in astandard PCR reaction (100 μl total volume) containing 100 ng ofFlt4-specific primers introducing SacI and PstI restriction sites, and 1unit of Taq DNA polymerase (Perkin Elmer Cetus). Two rounds of PCR wereperformed using 33 cycles per round (denaturation at 95° C. for 1minute, annealing at 55° C. for 2 minutes, and elongation at 72° C. for4 minutes). The PCR mixture was treated sequentially with the SacI andPstI restriction enzymes, and after purification with MagicPCR Preps(Promega), DNA fragments were subcloned into the pGEM3Zf(+) vector forsequencing (Promega). The sequence corresponded to the 5′ end of theFlt4s cDNA clone deposited in the Genbank Database as Accession No.X68203.

The sequence encoding the first 12 amino acid residues was added to theexpression construct by ligating an SphI-digested PCR fragment amplifiedusing reverse transcription-PCR of poly(A)⁺ RNA isolated from the HELcells. The forward primer had the following sequence: 5′-ACATGCATGCCACCATGCAG CGGGGCGCCG CGCTGTGCCT GCGACTGTGG CTCTGCCTGG GACTCCTGGA-3′(SEQ ID NO: 2) (SphI site underlined, translational start codon markedin bold). The translation start codon is immediately downstream from anoptimized Kozak consensus sequence. Kozak, Nucl. Acids Res., 15:8125-8148, 1987). The reverse primer, 5′-ACATGCATGC CCCGCCGGT CATCC-3′(SEQ ID NO: 3) (SphI site underlined), to the 5′ end of the S2.5fragment, thus replacing the unique SphI fragment of the S2.5 plasmid.The resulting vector was digested with EcoRI and ClaI and ligated to a138 bp PCR fragment amplified from the 0.6 kb EcoRI fragment (base pairs3789 to 4416 in the Genbank X68203 sequence) which encodes the 3′ end ofFlt4s shown in FIG. 1 of Pajusola et al., Cancer Res., 52:5738-5743(1992), using the oligonucleotides 5′-CGGAATTCCC CATGACCCCA AC-3′ (SEQID NO: 4) (forward primer, EcoRI site underlined) and 5′-CCATCGATGGATCCTACCTG AAGCCGCTTT CTT-3′ (SEQ ID NO: 5) (reverse primer, ClaI siteunderlined). The coding domain was completed by ligation of the 1.2 kbEcoRI fragment (base pairs 2535-3789 of the sequence found in Gen BankAcc. No. X68203) into the above construct. The complete cDNA wassubcloned as a HindIII-ClaI(blunted) fragment (this ClaI site was alsoincluded in the 3′ primer used to construct the 3′ end of the codingsequence) to the pLTRpoly expression vector reported in Mäkelä et al.,Gene, 118: 293-294 (1992) (Genbank accession number X60280, SEQ ID NO:37), incorporated by reference herein, using its HindIII-AccI(blunted)restriction sites.

The long form of Flt4 was produced by replacing the 3′-end of the shortform as follows: The 3′ region of the Flt4l cDNA was PCR-amplified usinga gene-specific oligonucleotide (SEQ ID NO: 7, see below) and a PGEM 3Zvector-specific (SP6 promoter) oligonucleotide 5′-ATTTAGGTGACACTATA-3′(SEQ ID NO: 6) as reverse and forward primers, respectively. Thetemplate for PCR was an Flt4l cDNA clone containing a 495 bp EcoRIfragment extending downstream of the EcoRI site at nucleotide 3789 ofthe Genbank X68203 sequence (the sequence downstream of this EcoRI siteis deposited as the Flt4 long form 3′ sequence having Genbank accessionnumber S66407 (SEQ ID NO: 38)). The gene-specific oligonucleotidecontains a BamHI restriction site located right after the end of thecoding region and has the following sequence:5′-CCATCGATGGATCCCGATGCTGCTTAGTAGCTGT-3′ (SEQ ID NO: 7) (BamI site isunderlined). The PCR product was digested with EcoRI and BamIH andtransferred in frame to the LTRFlt4s vector fragment from which thecoding sequences downstream of the EcoRI site at base pair 2535 (seesequence X68203) had been removed by EcoRI-BamHI digestion. Theresulting clone was designated pLTRFlt4l. Again, the coding domain wascompleted by ligation of the 1.2 kb EcoRI fragment (base pairs 2535-3789of sequence X68203) back into the resulting construct.

EXAMPLE 2 Production and Analysis of Flt4l Transfected Cells

NIH 3T3 cells (60% confluent) were co-transfected with 5 micrograms ofthe pLTRFlt4l construct and 0.25 micrograms of the pSV2neo vectorcontaining the neomycin phosphotransferase gene (Southern et al., J.Mol. Appl. Genet., 1:327 (1982)), using the DOTAP liposome-basedtransfection reagents (Boehringer-Mannheim, Mannheim, Germany). One dayafter transfection, the cells were transferred into selection mediacontaining 0.5 mg/ml geneticin (GIBCO, Grand Island, N.Y.). Colonies ofgeneticin-resistant cells were isolated and analyzed for expression ofthe Flt4 proteins. Cells were lysed in boiling lysis buffer containing3.3% SDS 125 mM Tris, pH 6.8. Protein concentrations of the samples weremeasured by the BCA method (Pierce, Rockford, Ill.). About 50 microgramsof protein from each lysate were analyzed for the presence of Flt4 by 6%SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblottingusing antisera against the carboxyl terminus of Flt4. Signals on Westernblots were revealed using the ECL method (Amersham).

For production of anti-Flt4 antiserum, the Flt4 cDNA fragment encodingthe 40 carboxy-terminal amino acid residues of the short form:NH2-PMTPTTYKG SVDNQTDSGM VLASEEFEQI ESRHRQESGFR-COOH (SEQ ID NO: 8) wascloned as a 657 bp EcoRI-fragment into the pGEX-1λT bacterial expressionvector (Pharmacia-LKB, Inc., Uppsala, Sweden) in frame with theglutathione-S-transferase coding region. The resultant GST-Flt4S fusionprotein was produced in E. coli and purified by affinity chromatographyusing a glutathione-Sepharose 4B column. The purified protein waslyophilized, dissolved in phosphate-buffered saline (PBS), mixed withFreund's adjuvant and used for immunization of rabbits at bi-weeklyintervals using methods standard in the art (Harlow et al., Antibodies:A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988)).Antisera were used, after the fourth booster immunization, forimmunoprecipitation of Flt4 from transfected cells. Cell clonesexpressing Flt4 were also used for ligand stimulation analysis.

EXAMPLE 3 Construction of a Flt4 EC Baculovirus Vector and Expressionand Purification of its Product

The construction of an Flt4 extracellular domain (EC) baculovirus vectoris schematically depicted in FIG. 3. The Flt4-encoding cDNA was preparedin both a long form and a short form, each being incorporated in avector under control of the Moloney murine leukemia virus LTR promoter.The nucleotide sequence of the short form of the Flt4 receptor isavailable from the Genbank database as Accession No. X68203 and thespecific 3′ segment of the long form cDNA is available under GenBankAccession No. S66407.

The ends of a cDNA segment encoding the Flt4 extracellular domain (EC)were modified as follows: The 3′ end of the Flt4 cDNA sequence (GenbankAccession Number X68203) which encodes the extracellular domain wasamplified using primer 1116 (5′-CTGGAGTCGACTTGGCGGACT-3′; SEQ ID NO:. 9,SalI site underlined) and primer 1315(5′-CGCGGATCCCTAGTGATGGTGATGGTGATGTCTACCTTCGATCATGCTGCCCTTAT CCTC-3′;(SEQ ID NO: 10, BamHI site underlined). The sequence at the 5′ end ofprimer 1315 is not complementary to the flt4 coding region. Inspectionof the sequence that is complementary to this region of prier 1315reveals in a 5′ to 3′ order, a stop codon, six contiguous histidinecodons (for subsequent chromatographic purification of the encodedpolypeptide using a Nc-NTA column; Qiagen, Hilden, Germany), and anadded BamHI site. The amplified fragment was digested with SalI andBamHI and used to replace a unique SalI-BamHI fragment in the LTRFlt4vector shown in FIG. 3. The SalI-BamHI fragment that was replacedencodes the Flt4 transmembrane and cytoplasmic domains. The result was amodified LTF Flt4 vector.

The 5′ end without the Flt4 signal sequence encoding region wasamplified by PCR using the primer 1335(5′-CCCAAGCTTGGATCCAAGTGGCTACTCCATGACC-3′; (SEQ ID NO: 11) the primercontains added HindIII (AAGCTT) and BamHI (GGATCC) restriction sites,which are underlined). The second primer used to amplify the regionencoding the Flt4 signal sequence was primer 13325′-GTTGCCTGTGATGTGCACCA-3′; SEQ ID NO: 12). The amplified fragment wasdigested with HindIII and SphI (the HindIII site (AAGCTT) is underlinedin primer 1335 and the SphI site is within the amplified region of theFlt4l cDNA). The resultant HindIII-SphI fragment was used to replace aHindIII-SphI fragment in the modified LTRFlt4l vector describedimmediately above (the HindIII site is in the 5′ junction of the Flt4insert with the pLTRpoly portion of the vector, the SphI site is in theFlt4 cDNA). The resultant Flt4EC insert was then ligated as a BamHIfragment into the BamHI site in the pVTBac plasmid described in Tessieret al., Gene 98:177-183 (1991), incorporated herein by reference. Therelative orientation of the insert was confirmed by partial sequencingso that the open reading frame of the signal sequence-encoding portionof the vector was adjacent to, and in frame with, the Flt4 coding regionsequence. The Flt4EC construct was transfected together with baculovirusgenomic DNA into SF-9 cells by lipofection. Recombinant virus waspurified, amplified and used for infection of High-Five cells(Invitrogen, San Diego, Calif.) using methods standard in the art. TheFlt4 extracellular domain (Flt4EC) was purified from the culture mediumof the infected High-Five cells using Ni-NTA affinity chromatographyaccording to manufacturer's instructions (Qiagen) for binding andelution of the 6×His tag encoded in the COOH-terminus of the recombinantFlt4 extracellular domain.

EXAMPLE 4 Isolation of an Flt4 Ligand from Conditioned Media

A human Flt4 ligand according to the invention was isolated from mediaconditioned by: a PC-3 prostatic adenocarcinoma cell line (ATCC CRL1435) in Harris F-12 Nutrient mixture (GIBCO) containing 7% fetal calfserum (FCS). The cells were grown according to the supplier'sinstructions. In order to prepare the conditioned media, confluent PC-3cells were cultured for 7 days in Ham's F-12 Nutrient mixture (GIBCO) inthe absence of fetal bovine serum (FBS). Medium was then cleared bycentrifugation at 10,000 g for 20 minutes. The medium was then screenedto determine its ability to induce tyrosine phosphorylation of Flt4 byexposure to NIH 3T3 cells which had been transfected with Flt4-encodingcDNA using the pLTRFlt4l vector. For receptor stimulation experiments,subconfluent NIH 3T3 cells were starved overnight in serum-free DMEMmedium (GIBCO) containing 0.2% bovine serum albumin (BSA). The cellswere stimulated with the conditioned media for 5 minutes, washed twicewith cold PBS containing 100 micromolar vanadate, and lysed in RIPAbuffer (10 mM Tris pH 7.5, 50 mM NaCl, 0.5% sodium deoxycholate, 0.5%Nonidet P40 (BDH, Poole, England), 0.1% SDS, 0.1 U/ml Aprotinin(Boehringer Mannheim), 1 mM vanadate) for receptor immunoprecipitationanalysis. The lysates were centrifuged for 20 minutes at 15,000×g. Thesupernatants were incubated for 2 hours on ice with 3 microliters of theantiserum against the Flt4 C-terminus described in Example 2. See alsoPajusola et al., Oncogene, 8:2931-2937 (1993), incorporated by referenceherein.

After a two hour incubation in the presence of anti-Flt4 antiserum,protein A-Sepharose (Pharmacia) was added and incubation was continuedfor 45 minutes with rotation. The immunoprecipitates were washed threetimes with the immunoprecipitation buffer and twice with 10 mM Tris, pH7.5, before analysis by SDS-PAGE. Polypeptides were transferred tonitrocellulose and analyzed by Western blotting using Flt4- orphosphotyrosine-specific antisera and the ECL method (Amersham Corp.).Anti-phosphotyrosine monoclonal antibodies (anti-PTyr; PY20) werepurchased from Transduction Laboratories (Lexington, Ky.). In somecases, the filters were restained with a second antibody afterstripping. The stripping of the filters was done for 30 minutes at 50°C. in 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7 withoccasional agitation.

As shown in FIG. 4, the PC-3 conditioned medium stimulated tyrosinephosphorylation of a 125 kD polypeptide when Flt4-expressing NIH 3T3cells were treated with the indicated preparations of media, lysed, andthe lysates were immunoprecipitated with anti-Flt4 antiserum followed bySDS-PAGE, Western blotting, and staining using anti-PTyr antibodies. Theresulting band was weakly phosphorylated upon stimulation withunconcentrated PC-3 conditioned medium (lane 2). The 125 kD bandcomigrated with the tyrosine phosphorylated, processed form of themature Flt4 from pervanadate-treated cells (compare lanes 2 and 7 ofFIG. 4, see also FIG. 5A). Comigration was confirmed upon restainingwith anti-Flt4 antibodies as is also shown in FIG. 5A (panel on theright). In order to show that the 125 kD polypeptide is not anon-specific component of the conditioned medium reactive withanti-phosphotyrosine antibodies, 15 microliters of conditioned mediumwere separated by SDS-PAGE, blotted on nitrocellulose, and the blot wasstained with anti-PTyr antibodies. No signal was obtained (FIG. 5B).Also, unconditioned medium failed to stimulate Flt4 phosphorylation, asshown in FIG. 4, lane 1.

FIG. 5C shows a comparison of the effects of PC-3 CM stimulation (+) onuntransfected (lanes 4 and 5), FGFR-4-transfected (lanes 8 and 9) andFlt4-transfected NIH 3T3 cells (lanes 1-3, 6 and 7). These resultsindicate that neither untransfected NIH 3T3 cells nor NIH 3T3 cellstransfected with FGFR-4 showed tyrosine phosphorylation of a protein ofabout 125 kD upon stimulation with the conditioned medium from PC-3cells. Analysis of stimulation by PC-3 CM pretreated withHeparin-Sepharose CL-6B (Pharmacia) for 2 hours at room temperature(lane 3) showed that the Flt4 ligand does not bind to heparin.

As shown in FIG. 4, lane 3, stimulating activity was considerablyincreased when the PC-3 conditioned medium was concentrated four-foldusing a Centricon-10 concentrator (Amicon). FIG. 4, lane 4, shows thatpretreatment of the concentrated PC-3 conditioned medium with 50microliters of the Flt4 extracellular domain coupled to CNBr-activatedsepharose CL-4B (Pharmacia; about 1 mg of Flt4EC domain/ml sepharoseresin) completely abolished Flt4 tyrosine phosphorylation. Similarpretreatment of the conditioned medium with unsubstituted sepharoseCL-4B did not affect stimulatory activity, as shown in FIG. 4, lane 5.Also, the flow through obtained after concentration, which containedproteins of less than 10,000 molecular weight, did not stimulate Flt4phosphorylation, as shown in FIG. 4, lane 6.

In another experiment, a comparison of Flt4 autophosphorylation intransformed NIH 3T3 cells expressing LTRFlt4l was conducted, usingunconditioned medium, medium from PC-3 cells expressing the Flt4 ligand,or unconditioned medium containing either 50 ng/ml of VEGF165 or 50ng/ml of PlGF-1. The cells were lysed, immunoprecipitated usinganti-Flt4 antiserum and analyzed by Western blotting usinganti-phosphotyrosine antibodies. As shown in FIG. 8, only the PC-3conditioned medium expressing the Flt4 ligand (lane Flt-4L) stimulatedFlt4 autophosphorylation.

The foregoing data show that PC-3 cells produce a ligand which binds tothe extracellular domain of Flt4 and activates this receptor.

EXAMPLE 5 Purification of the Flt4 Ligand

The ligand expressed by human PC-3 cells as characterized in Example 4was purified and isolated using a recombinantly-produced Flt4extracellular domain (Flt4EC) in affinity chromatography.

Two harvests of serum-free conditioned medium, comprising a total of 8liters, were collected from 500 confluent 15 cm diameter culture dishescontaining confluent layers of PC-3 cells. The conditioned medium wasclarified by centrifugation at 10,000×g and concentrated 80-fold usingan Ultrasette Tangential Flow Device (Filtron, Northborough, Mass.) witha 10 kD cutoff Omega Ultrafiltration membrane according to themanufacturer's instructions. Recombinant Flt4 extracellular domain wasexpressed in a recombinant baculovirus cell system and purified byaffinity chromatography on Ni-agarose (Ni-NTA affinity column obtainedfrom Qiagen). The purified extracellular domain was coupled toCNBr-activated Sepharose CL-4B at a concentration of 5 mg/ml and used asan affinity matrix for ligand affinity chromatography.

Concentrated conditioned medium was incubated with 2 ml of therecombinant Flt4 extracellular domain-Sepharose affinity matrix in arolling tube at room temperature for 3 hours. All subsequentpurification steps were at +4° C. The affinity matrix was thentransferred to a column with an inner diameter of 15 mm and washedsuccessively with 100 ml of PBS and 50 ml of 10 mM Na-phosphate buffer(pH 6.8). Bound material was eluted step-wise with 100 mM glycine-HCl,successive 6 ml elutions having pHs of 4.0, 2.4, and 1.9. Several 2 mlfractions of the eluate were collected in tubes containing 0.5 ml 1 MNa-phosphate (pH 8.0). Fractions were mixed immediately and dialyzed in1 mM Tris-HCl (pH 7.5). Aliquots of 75 μl each were analyzed for theirability to stimulate tyrosine phosphorylation of Flt4. Theultrafiltrate, 100 μl aliquots of the concentrated conditioned mediumbefore and after ligand affinity chromatography, as well as 15-foldconcentrated fractions of material released from the Flt4 extracellulardomain-Sepharose matrix during the washings were also analyzed for theirability to stimulate Flt4 tyrosine phosphorylation.

As shown in FIG. 6, lane 3, the concentrated conditioned medium inducedprominent tyrosine phosphorylation of Flt4 in transfected NIH 3T3 cellsoverexpressing Flt4. This activity was not observed in conditionedmedium taken after medium was exposed to the Flt4 Sepharose affinitymatrix described above (FIG. 6, lane 4). The specifically-boundFlt4-stimulating material was retained on the affinity matrix afterwashing in PBS, 10 mM Na-phosphate buffer (pH 6.8), and at pH 4.0 (FIG.6, lanes 5-7, respectively), and it was eluted in the first two 2 mlaliquots at pH 2.4 (lanes 8 and 9). A further decrease of the pH of theelution buffer did not cause release of additional Flt4-stimulatingmaterial (FIG. 6, lane 11). FIG. 6, lane 1 depicts a control whereinFlt4-expressing cells were treated with unconditioned medium; lane 2depicts the results following treatment of Flt4-expressing cells withthe ultrafiltrate fraction of conditioned medium containing polypeptidesof less than 10 kD molecular weight.

Small aliquots of the chromatographic fractions were concentrated in aSpeedVac concentrator (Savant, Farmingdale, N.Y.) and subjected toSDS-PAGE under reducing conditions with subsequent silver staining ofthe gel a standard technique in the art. As shown in FIG. 7, the majorpolypeptide, having a molecular weight of approximately 23 kD (reducingconditions), was detected in the fractions containing Flt4 stimulatingactivity (corresponding to lanes 8 and 9 in FIG. 6). That polypeptidewas not found in the other chromatographic fractions. On the other hand,all other components detected in the two active fractions were alsodistributed in the starting material and in small amounts in the otherwashing and eluting steps after their concentration. Similar resultswere obtained in three independent affinity purifications, indicatingthat the 23 kD polypeptide specifically binds to Flt4 and inducestyrosine phosphorylation of Flt4.

Fractions containing the 23 kD polypeptide were combined, dried in aSpeedVac concentrator and subjected to SDS-PAGE in a 12.5% gel. Theproteins from the gel were then electroblotted to Immobilon-P (PVDF)transfer membrane (Millipore, Marlborough, Mass.) and visualized bystaining of the blot with Coomassie Blue R-250. The region containingonly the stained 23 kD band was cut from the blot and subjected toN-terminal amino acid sequence analysis in a Prosite Protein SequencingSystem (Applied Biosystems, Foster City, Calif.). The data were analyzedusing a 610A Data Analysis System (Applied Biosystems). Analysisrevealed a single N-terminal sequence of NH₂-XEEIKFAAAHYNTEILK-COOH (SEQID NO: 13).

EXAMPLE 6 Construction of PC-3 Cell cDNA Library in a EukaryoticExpression Vector

Human poly(A)⁺ RNA was isolated from five 15 cm diameter dishes ofconfluent PC-3 cells by a single step method using oligo(dT) (Type III,Collaborative Biomedical Products, Becton-Dickinson Labware, Bedford,Mass.) cellulose affinity chromatography (Sambrook et al., 1989). Theyield was 70 micrograms. Six micrograms of the Poly(A)⁺ RNA were used toprepare an oligo(dT)-primed cDNA library in the mammalian expressionvector pcDNA I and the Librarian kit of Invitrogen according to theinstructions included in the kit. The library was estimated to containabout 10⁶ independent recombinants with an average insert size ofapproximately 1.8 kb.

EXAMPLE 7 Amplification of the Unique Nucleotide Sequence Encoding theFlt4 Ligand

Degenerate oligonucleotides were designed based on the N-terminal aminoacid sequence of the isolated human Flt4 ligand and were used as primersin a polymerase chain reaction (PCR) to amplify cDNA encoding the Flt4ligand from a PC-3 cell library. The overall strategy is schematicallydepicted in FIG. 9, where the different primers have been marked witharrows.

The PCR was carried out using 1 microgram of DNA from the amplified PC-3cDNA library and a mixture of 48 sense-strand primers present in equalproportions, the primer sequences collectively comprising the sequence5′-GCAGARGARACNATHAA-3′ (SEQ ID NO: 14) (wherein R is A or G, N is A,G,Cor T and H is A, C or T), encoding amino acid residues 2-6 (EETIK, SEQID NO: 15) and 384 antisense-strand primers present in equalproportions, the anti-sense strand primers collectively comprising thesequence 5′-GCAYTTNARDATYTCNGT-3′ (SEQ ID NO: 16) (wherein Y is C or Tand D is A, G or T), corresponding to amino acid residues 14-18 (TEILK,SEQ ID NO: 17). Three extra nucleotides (GCA) were added to the5′-terminus of each primer to increase annealing stability. Twosuccessive PCR runs were carried out using 1 U per reaction of DynaZyme(F-500L, Finnzymes, Espoo, Finland), a thermostable DNA polymerase, in abuffer supplied by the manufacturer (10 mM Tris-HCl, pH 8.8 at 25° C.,1.5 mM MgCl₂, 50 mM KCl, 0.1% Triton-X100), at an extension temperatureof 72° C. The first PCR run was carried out for 43 cycles. The firstthree cycles were run at an annealing temperature of 33° C. for 2minutes, and the remaining cycles were run at 42° C. for 1 minute.

The region of the gel containing a weak band of the expected size (57bp) was cut out from the gel and eluted. The eluted material wasreamplified for 30 cycles using the same primer pairs described above at42° C. for 1 minute. The amplified fragment was cloned into a pCR IIvector (Invitrogen) using the TA cloning kit (Invitrogen) and sequencedusing the radioactive dideoxynucleotide sequencing method of Sanger. Sixclones were analyzed and all six clones contained the sequence encodingthe expected peptide (amino acid residues 104-120 of the Flt4 ligandprecursor). Nucleotide sequence spanning the region from the thirdnucleotide of codon 6 to the third nucleotide of codon 13 (the extensionregion) was identical in all six clones: 5′-ATTCGCTGCAGCACACTACAAC-3′(SEQ ID NO: 18) and thus represented an amplified product from theunique sequence encoding part of the amino terminus of the Flt4 ligand.

EXAMPLE 8 Amplification of the 5′-end of the cDNA Encoding the Flt4Ligand

Based on the unique nucleotide sequence encoding the N-terminus of theisolated human Flt4 ligand, two pairs of nested primers were designed toamplify, in two nested PCR reactions, the complete 5′-end of thecorresponding cDNAs from one microgram of DNA of the above-describedPC-3 cDNA library. First, amplification was performed with an equalmixture of 4 primers collectively defining the sequence5′-TCNGTGTTGTAGTGTGCTG-3′ (SEQ ID NO: 19), which is the antisense-strandprimer corresponding to amino acid residues 9-15 (AAHYNTE, SEQ ID NO:20), and sense-strand primer 5′-TAATACGACTCACTATAGGG-3′ (SEQ ID NO: 21),corresponding to the T7 RNA promoter of the pcDNAI vector used forconstruction of the library. “Touchdown” PCR was used as disclosed inDon et al., Nucl. Acids Res., 19:4008 (1991), incorporated by referenceherein. The annealing temperature of the two first cycles was 62° C. andsubsequently the annealing temperature was decreased in every othercycle by 1° C. until a final temperature of 53° C. was reached, at whichtemperature 16 additional cycles were conducted. Annealing time was 1minute and extension at each cycle was conducted at 72° C. for 1 minute.Multiple amplified DNA fragments were obtained in the first reaction.The products of the first amplification (1 μl of a 1:100 dilution inwater) were used in the second amplification reaction employing a pairof nested primers comprising an antisense-strand primer5′-GTTGTAGTGTGCTGCAGCGAATTT-3′; SEQ ID NO: 22) encoding amino acidresidues 6-13 (KFAAAHYN, SEQ ID NO: 23) of the Flt4 ligand, and asense-strand primer (5′-TCACTATAGGGAGACCCAAGC-3′; SEQ ID NO: 24),corresponding to nucleotides 2179-2199 of the pcDNAI vector. Thesequences of these sense and antisense primers overlapped with the 3′ends of the corresponding primers used in the first PCR. “Touchdown” PCRwas carried out by decreasing the annealing temperature from 72° C. to66° C. and continuing with 18 additional cycles at 66° C. The annealingtime was 1 minute and extension at each cycle was carried out at 72° C.for 2 minutes. One major product of about 220 bp and three minorproducts of about 270 bp, 150 bp, and 100 bp were obtained.

The amplified fragment of approximately 220 bp was excised from anagarose gel, cloned into a pCRII vector using the TA cloning kit(Invitrogen), and sequenced. Three recombinant clones were analyzed andthey contained the sequence5′TCACTATAGGGAGACCCAAGCTTGGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGGTGGAATTCGACGAACTCATGACTGTACTCTACCCAGAATATTGGAAAATGTACAAGTGTCAGCTAAGGCAAGGAGGCTGGCAACATAACAGAGAACAGGCCAACCTCAACTCAAGGACAGAAGAGACTATAAAATTCGCTGCAGCACACTACAAC- 3′ (SEQ ID NO: 25). The beginning ofthe sequence represents the pcDNAI vector and the underlined sequencerepresents the amplified product of the 5′-end of the insert.

EXAMPLE 9 Amplification of the 3′-end of cDNA Encoding the Flt4 Ligand

Based upon the amplified 5′-sequence of the clones encoding the aminoterminus of the 23 kD human Flt4 ligand, two pairs of non-overlappingnested primers were designed to amplify the 3′-portion of theFlt-4-ligand-encoding cDNA clones. The sense-strand primer5′-ACAGAGAACAGGCCAACC-3′ (SEQ ID NO: 26), corresponding to nucleotides152-169 of the amplified 5′-sequences of the Flt4 ligand (SEQ ID NO:25), and antisense-strand primer 5′-TCTAGCATTTAGGTGACAC-3′ (SEQ ID NO:27) corresponding to nucleotides 2311-2329 of the pcDNAI vector wereused in a first “touchdown” PCR. The annealing temperature of thereaction was decreased 1° C. every two cycles from 72° C. to 52° C., atwhich temperature 15 additional cycles were carried out. The annealingtime was 1 minute and extension at each cycle was carried out at 72° C.for 3 minutes. DNA fragments of several sizes were obtained in the firstamplification. Those products were diluted 1:200 in water andreamplified in PCR using the second pair of primers:5′-AAGAGACTATAAAATTCGCTGCAGC-3′ (SEQ ID NO: 28) and5′-CCCTCTAGATGCATGCTCGA-3′ (SEQ ID NO: 29) (antisense-strand primercorresponding to nucleotides 2279-2298 of the pcDNAI vector). Two DNAfragments were obtained, having sizes of 1350 bp and 570 bp. Thosefragments were cloned into a pCRII vector and the inserts of the cloneswere sequenced. Both of these fragments were found to contain sequencesencoding an amino acid sequence homologous to the VEGF sequence.

EXAMPLE 10 Screening the PC-3 Cell cDNA Library Using the 5′ PCRFragment of Flt4 Ligand cDNA

A 219 bp 5′-terminal fragment of human Flt4 ligand cDNA was amplified byPCR using the 5′ PCR fragment described above and primers5′-GTTGTAGTGTGCTGCAGCGAATTT-3′ (antisense-strand primer, SEQ ID NO: 30)and 5′-TCACTATAGGGAGACCCAAGC-3′ (SEQ ID NO: 31) (sense-primercorresponding to nucleotides 2179-2199 of the pcDNAI vector). Theamplified product was subjected to digestion with EcoRI (BoehringerMannheim) to remove the portion of the DNA sequence amplified from thepcDNAI vector and the resulting 153 bp fragment encoding the 5′ end ofthe Flt4 ligand was labeled with [³²P]-dCTP using the Klenow fragment ofE. coli DNA polymerase I (Boehringer Mannheim). That fragment was usedas a probe for hybridization screening of the amplified PC-3 cell cDNAlibrary.

Filter replicas of the library were hybridized with the radioactivelylabeled probe at 42° C. for 20 hours in a solution containing 50%formamide, 5×SSPE, 5×Denhardt's solution, 0.1 % SDS and 0.1 mg/mldenatured salmon sperm DNA. Filters were washed twice in 1×SSC, 0.1% SDSfor 30 minutes at room temperature, then twice for 30 minutes at 65° C.and exposed overnight.

On the basis of autoradiography, 10 positive recombinant bacterialcolonies hybridizing with the probe were chosen from the library.Plasmid DNA was purified from these colonies and analyzed by EcoRI andNotI digestion and agarose gel electrophoresis followed by ethidiumbromide staining. The ten plasmid clones were divided into three groupson the basis of the presence of insert sizes of approximately 1.7, 1.9and 2.1 kb, respectively. Inserts of plasmids from each group weresequenced using the T7 oligonucleotide as a primer and walking primersfor subsequent sequencing reactions.

Sequence analysis showed that all clones contain the open reading frameencoding the NH2-terminal sequence of the 23 kD human Flt4 ligand.Dideoxy sequencing was continued using walking primers in the downstreamdirection. A complete human cDNA sequence and deduced amino acidsequence from a 2.1 kb clone is set forth in FIG. 9. A putative cleavagesite of a “prepro” leader sequence is located between resides 102 and103 of SEQ ID NO: 33. When compared with sequences in the GenBankDatabase, the predicted protein product of this reading frame was foundto be homologous with the predicted amino acid sequences of thePDGF/VEGF family of growth factors, as shown in FIGS. 10A-10C.

Plasmid pFLT4-L, containing the 2.1 kb human cDNA clone in pcDNAIvector, has been deposited with the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md. 20852 as accession number 97231.

EXAMPLE 11 Stimulation of Flt4 Autophosphorylation by the ProteinProduct of the Flt4 Ligand Vector

The 2.1 kb human cDNA insert of plasmid pFlt4-L, which contains the openreading frame encoding the sequence shown in SEQ ID NO: 32-33 (humanVEGF-C, see below), was cut out from the pcDNAI vector using HindIII andNotI restriction enzymes, isolated from a preparative agarose gel, andligated to the corresponding sites in the pREP7 expression vector(Invitrogen). The pREP7 vector containing the pFlt4-L insert wastransfected into 293-EBNA cells (Invitrogen) using the calcium phosphatetransfection method (Sambrook et al., 1989). About 48 hours aftertransfection the medium of the transfected cells was changed to DMEMmedium lacking fetal calf serum and incubated for 36 h. The conditionedmedium was then collected, centrifuged at 5000×g for 20 minutes, thesupernatant was concentrated 5-fold using Centriprep 10 (Amicon) andused to stimulate NIH 3T3 cells expressing LTRFlt4(the Flt4 receptor),as in Example 4. The cells were lysed, immunoprecipitated usinganti-Flt4 antiserum and analyzed by Western blotting usinganti-phosphotyrosine antibodies.

As can be seen from FIG. 11, lanes 1 and 3, the conditioned medium fromtwo different dishes of the transfected cells stimulated Flt4autophosphorylation in comparison with the medium from mock-transfectedcells, which gave only background levels of phosphorylation of the Flt4receptor (lane 2). When the concentrated conditioned medium waspre-absorbed with 20 microliters of a slurry of Flt4EC domain coupled toSepharose (see example 4), no phosphorylation was obtained (lane 4),showing that the activity responsible for Flt4 autophosphorylation wasindeed the Flt4 ligand. Thus, these results demonstrate that anexpression vector having an approximately 2.1 kb insert and containingan open reading frame as shown in SEQ ID NO: 32 is expressed as abiologically active Flt4 ligand (VEGF-C) in transfected cells. Thesequence encoded by that open reading frame is shown in SEQ ID NO: 33.

The deduced molecular weight of a polypeptide consisting of the completeamino acid sequence in SEQ ID NO: 33 (residues 1 to 419) is 46,883. Thededuced molecular weight of a polypeptide consisting of amino acidresidues 103 to 419 of SEQ ID NO: 33 is 35,724. The Flt4 ligand purifiedfrom PC-3 cultures had an observed molecular weight of about 23 kD asassessed by SDS-PAGE under reducing conditions. Thus, it appears thatthe Flt4 ligand mRNA is translated into a precursor polypeptide, fromwhich the mature ligand is derived by proteolytic cleavage. Also, theFlt4 ligand may be glycosylated at three putative N-linked glycosylationsites conforming to the consensus which can be identified in the deducedFlt4 ligand amino acid sequence (N-residues underlined in FIGS.10A-10C).

The carboxyl terminal amino acid sequences, which increase the predictedmolecular weight of the Flt4 ligand subunit in comparison with otherligands of this family, show a pattern of spacing of cysteine residuesreminiscent of the Balbiani ring protein 3 (BR3P) sequence (Dignam etal., Gene, 88:133-140 (1990)), as depicted schematically in FIG. 9. Sucha sequence may encode an independently folded domain present in a Flt4ligand precursor and it may be involved, for example, in the regulationof secretion, solubility, stability, cell surface localization oractivity of the Flt4 ligand. Interestingly, at least one cysteine motifof the BR3P type is also found in the VEGF carboxy terminal amino acidsequences.

Thus, the Flt4 ligand mRNA appears first to be translated into aprecursor from the mRNA corresponding to the cDNA insert of plasmidFLT4-L, from which the mature ligand is derived by proteolytic cleavage.To define the mature Flt4 ligand polypeptide, one first expresses thecDNA clone (which is deposited in the pcDNAI expression vector) incells, such as COS cells. One uses antibodies generated against encodedpeptides, fragments thereof, or bacterial Flt4 fusion proteins, such asa GST-fusion protein, to raise antibodies against the VEGF-homologousdomain and the amino- and carboxyl-terminal propeptides of Flt4 ligand.One then follows the biosynthesis and processing of the Flt4 ligand inthe transfected cells by pulse-chase analysis using radioactive cysteinefor labelling-of the cells, immunoprecipitation and gel electrophoresis.Using antibodies against the three domains of the product encoded by thecDNA insert of plasmid FLT4-L, material for radioactive ornonradioactive amino-terminal sequence analysis is isolated. Thedetermination of the amino-terminal sequence of the mature VEGF-Cpolypeptide allows for identification of the amino-terminal proteolyticprocessing site. The determination of the amino-terminal sequence of thecarboxyl-terminal propeptide will give the carboxyl-terminal processingsite. This is confirmed by site-directed mutagenesis of the amino acidresidues adjacent to the cleavage sites, which would prevent thecleavage.

On the other hand, the Flt4 ligand is characterized by progressive 3′deletions in the 3′ coding sequences of the Flt4 ligand precursor clone,introducing a stop codon resulting in carboxy-terminal truncations ofits protein product. The activities of such truncated forms are assayedby, for example, studying Flt4 autophosphorylation induced by thetruncated proteins when applied to cultures of cells, such as NIH 3T3cells expressing LTRFlt4. By extrapolation from studies of the structureof the related platelet derived growth factor (PDGF, Heldin et al.,Growth Factors, 8:245-252 (1993)) one determines that the regioncritical for receptor activation by the Flt4 ligand is contained withinits first approximately 180 amino acid residues of the secreted VEGF-Cprotein lacking the putative 102 amino acid prepro leader, andapparently within the first approximately 120 amino acid residues.

On the other hand, the difference between the molecular weights observedfor the purified ligand and deduced from the open reading frame of theFlt4 precursor clone may be due to the fact that the soluble ligand wasproduced from an alternatively spliced mRNA which would also be presentin the PC-3 cells, from which the isolated ligand was derived. Toisolate such alternative cDNA clones one uses cDNA fragments of thedeposited clone and PCR primers made according to the sequence providedas well as techniques standard in the art to isolate or amplifyalternative cDNAs from the PC-3 cell cDNA library. One may also amplifyusing reverse transcription (RT)-PCR directly from the PC-3 mRNA usingthe primers provided in the sequence of the cDNA insert of plasmidFLT4-L. Alternative cDNA sequences are determined from the resultingcDNA clones. One can also isolate genomic clones corresponding to theFlt4 ligand mRNA transcript from a human genomic DNA library usingmethods standard in the art and to sequence such clones or theirsubcloned fragments to reveal the corresponding exons. Alternative exonscan then be identified by a number of methods standard in the art, suchas heteroduplex analysis of cDNA and genomic DNA, which are subsequentlycharacterized.

EXAMPLE 12 Expression of the Gene Encoding VEGF-C in Human Tumor CellLines

Expression of transcripts corresponding to the Flt4 ligand (VEGF-C) wasanalyzed by hybridization of Northern blots containing isolated poly(A)⁺RNA from HT-1080 and PC-3 human tumor cell lines. The probe was theradioactively labelled insert of the 2.1 kb cDNA clone (pFlt4-L/VEGF-C)specific activity 10⁸-10⁹ cpm/mg of DNA). The blot was hybridizedovernight at 42° C. using 50% formamide, 5×SSPE buffer, 2% SDS,10×Denhardt's solution, 100 mg/ml salmon sperm DNA and 1×10⁶ cpm of thelabelled probe/ml. The blot was washed at room temperature for 2×30minutes in 2×SSC containing 0.05% SDS, and then for 2×20 min at 52° C.in 0.1×SSC containing 0.1% SDS. The blot was then exposed at −70° C. forthree days using intensifying screens and Kodak XAR film. Both celllines expressed an Flt4 ligand mRNA of about 2.4 kb, as well as VEGF andVEGF-B mRNAs (FIG. 12).

EXAMPLE 13 VEGF-C Chains Are Proteolytically Processed afterBiosynthesis and Disulfide Linked

The predicted molecular mass of a secreted human VEGF-C polypeptide, asdeduced from the VEGF-C open reading frame, is 46,883 kD, suggestingthat VEGF-C mRNA may be first translated into a precursor, from whichthe ligands of 21/23 kD and 29/32 kD are derived by proteolyticcleavage.

This possibility was explored by metabolic labelling of 293 EBNA cellsexpressing VEGF-C. Initially, 293 EBNA cells were transfected with theVEGF-C construct. Expression products were labeled by the addition of100 μCi/ml of Pro-mix™ L-[³⁵S] in vitro cell labelling mix (Amersham) tothe culture medium devoid of cysteine and methionine. After two hours,the cell layers were washed twice with. PBS and the medium was thenreplaced with DMEM-0.2% BSA. After 1, 3, 6, 12 and 24 hours ofsubsequent incubation, the culture medium was collected, clarified bycentrifugation, and concentrated, and human VEGF-C was bound to 30 μl ofa slurry of Flt4EC-Sepharose overnight at +4° C., followed by threewashes in PBS, two washes in 20 mM Tris-HCl (pH 7.5), alkylation,SDS-PAGE and autoradiography. Alkylation was carried out by treatment ofthe samples with 10 mM 1,4 Dithiothreitol (Boehringer-Mannheim,Mannheim, Germany) for one hour at 25° C., and subsequently with 30 mMiodoacetamide (Fluka, Buchs, Switzerland).

These experiments demonstrated that a putative precursor polypeptide of32 kD apparent molecular mass was bound to the Flt4EC affinity matrixfrom the conditioned medium of metabolically labelled cells transfectedwith a human VEGF-C expression vector (FIG. 13A). Increased amounts of a23 kD receptor binding polypeptide accumulated in the culture mediumduring a subsequent chase period of three hours, but not thereafter(lanes 2-4 and data not shown), suggesting that the 23 kD form isproduced by proteolytic processing, which is cell-associated andincomplete, at least in the transiently transfected cells. The arrows inFIG. 13A indicate the 32 kD and 23 kD polypeptides of secreted VEGF-C.Subsequent experiments showed that the 32 kD VEGF-C form contains twocomponents migrating in the absence of alkylation as polypeptides of 29and 32 kD (FIGS. 21-23).

In a related experiment, human VEGF-C isolated using Flt4EC-Sepharoseafter a 4 h continuous metabolic labelling was analyzed bypolyacrylamide gel electrophoresis in nonreducing conditions (FIG. 13B).Higher molecular mass forms were observed under nonreducing conditions,suggesting that the VEGF-C polypeptides can form disulfide-linked dimersand/or multimers (arrows in FIG. 13B).

EXAMPLE 14 Stimulation Of VEGFR-2 Autophosphorylation by VEGF-C

Conditioned medium (CM) from 293 EBNA cells transfected with the humanVEGF-C vector also was used to stimulate porcine aortic endothelial(PAE) cells expressing VEGFR-2. Pajusola et al., Oncogene, 9:3545-55(1994); Waltenberger et al., J. Biol. Chem., 269:26988-26995 (1994). Thecells were lysed and immunoprecipitated using VEGFR-2-specific antiserum(Waltenberger et al., 1994).

PAE-KDR cells (Waltenberger et al., 1994) were grown in Ham's F12medium-10% fetal calf serum (FCS). Confluent NIH 3T3-Flt4 cells orPAE-KDR cells were starved overnight in DMEM or Ham's F12 medium,respectively, supplemented with 0.2% bovine serum albumin (BSA), andthen incubated for 5 min with the analyzed media. Recombinant human VEGF(R&D Systems) and PDGF-BB, functional as stimulating agents, were usedas controls. The cells were washed twice with ice-cold Tris-BufferedSaline (TBS) containing 100 mM sodium orthovanadate and lysed in RIPAbuffer-containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.1 U/mlaprotinin and 1 mM sodium orthovanadate. The lysates were sonicated,clarified by centrifugation at 16,000×g for 20 min and incubated for 3-6h on ice with 3-5 μl of antisera specific for Flt4 (Pajusola et al.,1993), VEGFR-2 or PDGFR-β (Claesson-Welsh et al., J. Biol. Chem.264:1742-1747 (1989); Waltenberger et al., 1994). Immunoprecipitateswere bound to protein A-Sepharose, washed three times with RIPA buffercontaining 1 mM PMSF, 1 mM sodium orthovanadate, washed twice with 10 mMTris-HCl (pH 7.4), and subjected to SDS-PAGE using a 7% gel.Polypeptides were transferred to nitrocellulose by Western blotting andanalyzed using PY20 phosphotyrosine-specific monoclonal antibodies(Transduction Laboratories) or receptor-specific antiserum and the ECLdetection method (Amersham Corp.).

The results of the experiment are presented in FIGS. 14A and 14B. Asshown in FIG. 14A, PAE cells expressing VEGFR-2 were stimulated with 10-or 2-fold concentrated medium from mock-transfected 293-EBNA cells(lanes 1 and 2), or with 2-, 5- or 10-fold concentrated medium from293-EBNA cell cultures expressing the recombinant VEGF-C (lanes 3-6).VEGFR-2 was immunoprecipitated with specific antibodies and analyzed bySDS-PAGE and Western blotting using phosphotyrosine antibodies. Forcomparison, the stimulations were carried out with non-conditionedmedium containing 50 ng/ml of purified recombinant VEGF (lanes 7 and 8).Lanes 6 and 7 show stimulation with VEGF-C- or VEGF-containing mediapretreated with Flt4EC. As depicted in FIG. 14B, PDGFR-β-expressing NIH3T3 cells were stimulated with non-conditioned medium (lane 1), 5-foldconcentrated CM from mock-transfected (lane 2) or VEGF-C-transfected(lanes 3 and 4) cells, or with non-conditioned medium containing 50ng/ml of recombinant human PDGF-BB (lane 5). Medium containing VEGF-Cwas also pretreated with recombinant Flt4EC (lane 4). PDGFR-β wasimmunoprecipitated with specific antibodies and analyzed by SDS-PAGE andWestern blotting using phosphotyrosine antibodies with subsequentstripping and reprobing of the membrane with antibodies specific forPDGFR-β.

Referring again to FIG. 14A, a basal level of tyrosine phosphorylationof VEGFR-2 was detected in cells stimulated by CM from themock-transfected cells. A further concentration of this medium resultedin only a slight enhancement of VEGFR-2 phosphorylation (lanes 1 and 2).CM containing recombinant VEGF-C stimulated tyrosine autophosphorylationof VEGFR-2 and the intensity of the autophosphorylated polypeptide bandwas increased upon concentration of the VEGF-C CM (lanes 3-5).Furthermore, the stimulating effect was abolished after pretreatment ofthe medium with the Flt4EC affinity matrix (compare lanes 1, 5 and 6).The maximal effect of VEGF-C in this assay was comparable to the effectof recombinant VEGF added to unconditioned medium at concentration of 50ng/ml (lane 8). Pretreatment of the medium containing VEGF with Flt4ECdid not abolish its stimulating effect on VEGFR-2 (compare lanes 7 and8). These results suggest that the VEGF-C expression vector encodes aligand not only for Flt4 (VEGFR-3), but also for VEGFR-2.

In order to further confirm that the stimulating effect of VEGF-C ontyrosine phosphorylation of VEGFR-3 and VEGFR-2 was receptor-specific,we analyzed the effect of VEGF-C on tyrosine phosphorylation of PDGFreceptor β (PDGFR-β) which is abundantly expressed on fibroblasticcells. As can be seen from FIG. 14B, a weak tyrosine phosphorylation ofPDGFR-β was detected upon stimulation of Flt4-expressing NIH 3T3 cellswith CM from the mock-transfected cells (compare lanes 1 and 2). Asimilar low level of PDGFRβ phosphorylation was observed when the cellswere incubated with CM from the VEGF-C transfected cells, with orwithout prior treatment with Flt4EC (lanes 3 and 4). In contrast, theaddition of 50 ng/ml of PDGF-BB induced a prominent tyrosineautophosphorylation of PDGFRβ (lane 5).

EXAMPLE 15 VEGF-C Stimulates Endothelial Cell Migration in Collagen Gel

CM from cell cultures transfected with the VEGF-C expression vector wasplaced in a well made in collagen gel and used to stimulate themigration of bovine capillary endothelial (BCE) cells in thethree-dimensional collagen gel as follows.

BCE cells (Folkman et al., Proc. Natl. Acad. Sci. (USA), 76:5217-5221(1979) were cultured as described in Pertovaara et al., J. Biol. Chem.,269:6271-74 (1994). The collagen gels were prepared by mixing type Icollagen stock solution (5 mg/ml in 1 mM HCl) with an equal volume of2×MEM and 2 volumes of MEM containing 10% newborn calf serum to give afinal collagen concentration of 1.25 mg/ml. The tissue culture plates (5cm diameter) were coated with about 1 mm thick layer of the solution,which was allowed to polymerize at 37° C. BCE cells were seeded on topof this layer. For the migration assays, the cells were allowed toattach inside a plastic ring (1 cm diameter) placed on top of the firstcollagen layer. After 30 min., the ring was removed and unattached cellswere rinsed away. A second layer of collagen and a layer of growthmedium (5% newborn calf serum (NCS)), solidified by 0.75% low meltingpoint agar (FMC BioProducts, Rockland, Me.), were added. A well (3 mmdiameter) was punched through all the layers on both sides of the cellspot at a distance of 4 mm, and the sample or control media werepipetted daily into the wells. Photomicrographs of the cells migratingout from the spot edge were taken after six days through an Olympus CK 2inverted microscope equipped with phase-contrast optics. The migratingcells were counted after nuclear staining with the fluorescent dyebisbenzimide (1 mg/ml, Hoechst 33258, Sigma).

FIG. 15A depicts a comparison of the number of cells migrating atdifferent distances from the original area of attachment towards wellscontaining media conditioned by the non-transfected (control) ortransfected (mock; VEGF-C; VEGF) cells, 6 days after addition of themedia. The number of cells migrating out from the original ring ofattachment was counted in five adjacent 0.5 mm×0.5 mm squares using amicroscope ocular lens grid and 10×magnification. Cells migratingfurther than 0.5 mm were counted in a similar way by moving the grid in0.5 mm steps. The experiments were carried out twice with similarresults, and medium values from the one of the experiments are presentedwith standard error bars. The photographs in FIGS. 15B and 15C depictphase-contrast microscopy and fluorescent microscopy of the nuclearstaining of BCE cells migrating towards the wells containing mediaconditioned by the mock-transfected cells or by VEGF-C-transfectedcells. The areas shown is approximately 1 mm×1.5 mm, and arrows indicatethe borders of the original ring of attachment.

After 6 days of treatment, the cultures were stained and cells atdifferent distances outside of the original ring of attachment werecounted using fluorescent nuclear staining and detection with afluorescence microscope equipped with a grid. A comparison of thenumbers of migrating cells in successive 0.5 mm×0.5 mm areas is shown inFIG. 15A. As can be seen from the columns, VEGF-C-containing CMstimulated cell migration more than medium conditioned by thenon-transfected or mock-transfected cells but less than medium fromcells transfected with a VEGF expression vector. An example of typicalphase contrast and fluorescent microscopic fields of cultures stimulatedwith medium from mock-transfected or VEGF-C transfected cells is shownin FIGS. 15B and 15C. Daily addition of 1 ng of FGF2 into the wellsresulted in the migration of approximately twice the number of cellswhen compared to the stimulation by CM from VEGF-transfected cells.

EXAMPLE 16 VEGF-C is Expressed In Multiple Tissues

Northern blots containing 2 micrograms of isolated poly(A)⁺ RNA frommultiple human tissues (blot from Clontech Laboratories, Inc., PaloAlto, Calif.) were probed with radioactively labelled insert of the 2.1kb VEGF-C cDNA clone. Northern blotting and hybridization analysisshowed that the 2.4 kb RNA and: smaller amounts of a 2.0 kb mRNA areexpressed in multiple human tissues, most prominently in the heart,placenta, muscle, ovary and small intestine (FIG. 16A). Very littleVEGF-C RNA was seen in the brain, liver or thymus and peripheral bloodleukocytes (PBL) appeared negative. A similar analysis of RNA from humanfetal tissues (FIG. 16B) shows that VEGF-C is highly expressed in thekidney and lung and to a lesser degree in the liver, while essentiallyno expression is detected in the brain. Interestingly, VEGF expressioncorrelates with VEGF-C expression in these tissues, whereas VEGF-B ishighly expressed in all tissues analyzed.

EXAMPLE 17 The VEGF-C Gene Localizes to Chromosome 4q34

A DNA panel of 24 interspecies somatic cell hybrids, which had retainedone or two human chromosomes, was used for the chromosomal localizationof the VEGF-C gene (Bios Laboratories, Inc., New Haven, Conn.). Primerswere designed to amplify an about 250 bp fragment of the VEGF-C genefrom somatic cell hybrid DNA. The primers and conditions for polymerasechain reaction (PCR) were 5′-TGAGTGATIIGTAGCTGCTGTG-3′ (forward) [SEQ IDNO: 34] and 5′-TATTGCAGCAACCCCCACATCT-3′ (reverse) [SEQ ID NO: 35] forVEGF-C (94° C., 60s/62° C., 45s/72° C., 60s). The PCR products wereevaluated by electrophoresis in 1% agarose gels and visualized byethidium bromide staining in ultraviolet light. [α-³²P]-dCTP-labelledcDNA inserts of a plasmid representing the complete VEGF-C coding domainwas used as a probe in Southern blotting and hybridization analysis ofthe somatic cell hybrid DNAs as instructed by the supplier (BiosLaboratories).

The cell lines for fluorescence in situ hybridization (FISH) wereobtained from the American Type Culture Collection (Rockville, Md.).Purified DNA from P1 clones 7660 and 7661 (VEGF-C) (Genome Systems,Inc., St. Louis, Mo.) were confirmed positive by Southern blotting ofEcoRI-digested DNA followed by hybridization with the VEGF-C cDNA. TheP1 clones were then labelled by nick translation either withbiotin-11-dUTP, biotin-14-ATP (Sigma Chemical Co., St. Louis, Mo.) ordigoxigenin 11-dUTP (Boehringer Mannheim GmbH, Mannheim, Germany)according to standard protocols. PHA-stimulated peripheral bloodlymphocyte cultures were treated with 5-bromodeoxyuridine (BrdU) at anearly replicating phase to induce G-banding. See Takahashi et al., HumanGenet., 86:14-16 (1995); Lemieux et al., Cytogenet. Cell Genet., 59:311-12 (1992). The FISH procedure was carried out in 50% formamide, 10%dextran sulphate in 2×SSC using well-known procedures. See e.g.,Rytkönnen et al., Cytogenet Cell Genet., 68:61-63 (1995); Lichter etal., Proc. Natl. Acad. Sci. (USA), 85:9664-68 (1988). Repetitivesequences were suppressed with 50-fold excess of Cot-1 DNA (BRL,Gaithersburg, Md.) compared with the labeled probe. Specifichybridization signals were detected by incubating the hybridized slidesin labelled antidigoxigenin antibodies, followed by counterstaining with0.1 mmol/L 4,6-diamino-2-phenylindole. Probe detection for two-colorexperiments was accomplished by incubating the slides in fluoresceinisothiocyanate (FITC)-anti-digoxigenin antibodies (Sigma Chemical Co.)and Texas red-avidin (Vector Laboratories, Burlingame, Calif.) orrhodamine-anti-digoxigenin and FITC-avidin.

Multi-color digital image analysis was used for acquisition, display andquantification of hybridization signals of metaphase chromosomes. Thesystem contains a PXL camera (Photometrics Inc., Tucson, Ariz.) attachedto a PowerMac 7100/Av workstation. IPLab software controls the cameraoperation, image acquisition and Ludl Filter wheel. At least 50 nucleiwere scored. Overlapping nuclei and clusters of cells were ignored. Aslide containing normal lymphocyte metaphase spreads and interphasenuclei was included in each experiment to control for the efficiency andspecificity of the hybridization.

In order to determine the chromosomal localization of the human VEGF-Cgene, DNAs from human rodent somatic cell hybrids containing definedsets of human chromosomes were analyzed by Southern blotting andhybridization with the VEGF-C cDNA probe. Among 24 DNA samples on thehybrid panel, representing different human chromosomes, human-specificsignals were observed only in hybrids which contained human chromosome4. The results were confirmed by PCR of somatic cell hybrid DNA usingVEGF-C specific primers, where amplified bands were obtained only fromDNAs containing human chromosome 4.

A genomic P1 plasmid for VEGF-C was isolated using specific primers andPCR and verified by Southern blotting and hybridization using a VEGF-Cspecific cDNA probe. The chromosomal localization of VEGF-C was furtherstudied using metaphase FISH. Using the P1 probe for VEGF-C in FISH aspecific hybridization to the 4q34 chromosomal band was detected in 40out of 44 metaphases (FIG. 17). Double-fluorochrome hybridization usinga cosmid probe specific for the aspartylglucosaminidase (AGA) geneshowed that VEGF-C is located just proximal to the AGA gene previouslymapped to the 4q34-35 chromosomal band.

Biotin labelled VEGF-C P1 and digoxigenin labeled AGA cosmid probes werehybridized simultaneously to metaphase chromosomes. This experimentdemonstrated that the AGA gene is more telomerically located than theVEGF-C gene. The foregoing example demonstrates the utility ofpolynucleotides of the invention as chromosomal markers.

EXAMPLE 18 Effect of Glucose Concentration and Hypoxia on VEGF, VEGF-Band VEGF-C mRNA Levels in C6 Glioblastoma Cells

Confluent cultures of C6 cells (ATCC CCL 107) were grown on 10 cmdiameter tissue culture plates containing 2.5 ml of DMEM and 5% fetalcalf serum plus antibiotics. The cultures were exposed for 16 hours tonormoxia in a normal cell culture incubator containing 5% CO₂ (FIG. 18:lanes marked −) or hypoxia (FIG. 18: lanes marked +) by closing theculture plates in an airtight glass chamber and burning a piece of woodinside until the flame was extinguished due to lack of oxygen.Polyadenylated RNA was isolated (as in the other examples), and 8micrograms of the RNA was electrophoresed and blot-hybridized with amixture of the VEGF, VEGF-B and VEGF-C probes (see FIG. 12). The resultsshow that hypoxia strongly induces VEGF (VEGF-A) mRNA expression(compare lanes − and +), both in low and high glucose, but has nosignificant effect on the VEGF-B mRNA levels. The VEGF-C mRNA isolatedfrom hypoxic cells runs slightly faster in gel electrophoresis and anextra band of faster mobility can be seen below the upper mRNA band.This observation suggests that hypoxia affects VEGF-C RNA processing.One explanation for this observation is that VEGF-C mRNA splicing isaltered, affecting the VEGF-C open reading frame and resulting in analternative VEGF-C protein being produced by hypoxic cells. Suchalternative forms of VEGF-C and VEGF-C-encoding polynucleotides arecontemplated as an aspect of the invention. This data indicatesscreening and diagnostic utilities for polynucleotides and polypeptidesof the invention, such as methods whereby a biological sample isscreened for the hypoxia-induced form of VEGF-C and/or VEGF-C mRNA. Thedata further suggests a therapeutic indication for antibodies and/orother inhibitors of the hypoxia-induced form of VEGF-C or the normalform of VEGF-C.

EXAMPLE 19 Pulse-chase Labeling and Immunoprecipitation of VEGF-CPolypeptides from 293 Cells Transfected with VEGF-C Expression Vector

The following VEGF-C branched amino-terminal peptide, designated PAM126,was synthesized for production of anti-VEGF-C antiserum:

NH₂-E-E-T-I-K-F-A-A-A-H-Y-N-T-E-I-L-K-COOH (SEQ ID NO: 39). Inparticular, PAM126 was synthesized as a branched polylysine structureK3PA4 having four peptide acid (PA) chains attached to two availablelysine (K) residues. The synthesis was performed on a 433A PeptideSynthesizer (Applied Biosystems) using Fmoc-chemistry and TentaGel S MAPRAM10 resin mix (RAPP Polymere GmbH, Tubingen, Germany), yielding bothcleavable and resin-bound peptides. The cleavable peptide was purifiedvia reverse phase HPLC and was used together with the resin-boundpeptide in immunizations. The correctness of the synthesis products wereconfirmed using mass-spectroscopy (Lasermatt).

The peptide was dissolved in phosphate buffered saline (PBS), mixed withFreund's adjuvant, and used for immunization of rabbits at bi-weeklyintervals using methods standard in the art (Harlow and Lane,Antibodies, a laboratory manual, Cold Spring Harbor Laboratory Press(1988)). Antisera obtained after the fourth booster immunization wasused for immunoprecipitation of VEGF-C in pulse-chase experiments, asdescribed below.

For pulse-chase analysis, 293 cells transfected with a VEGF-C expressionvector (i.e., the FLT4-L cDNA inserted into the pREP7 expression vectoras described above) were incubated for 30 minutes in methionine-free,cysteine-free, serum-free DMEM culture medium at 37° C. The medium wasthen changed, and 200 μCi of ³⁵S-methionine and ³⁵5-cysteine (Promix,Amersham, Buckinghamshire, England) was added. The cell layers wereincubated in this labeling medium for two hours, washed with PBS, andincubated for 0, 15, 30, 60, 90, 120, or 180 minutes in serum-free DMEM(chase). After the various chase periods, the medium was collected, thecells were again washed two times in PBS, and lysed inimmunoprecipitation buffer. The VEGF-C polypeptides were analyzed fromboth the culture medium and from the cell lysates byimmunoprecipitation, using the VEGF-C-specific antiserum raised againstthe NH₂-terminal peptide (PAM126) of the 23 kD VEGF-C form.Immunoprecipitated polypeptides were analyzed via SDS-PAGE followed byautoradiography.

Referring to FIG. 19, the resultant autoradiograms demonstrate thatimmediately after a 2 hour labeling (chase time 0), the VEGF-Cvector-transfected cells contained a radioactive 55 kD polypeptide band,which is not seen in mock-transfected cells (M). This 55 kD polypeptideband gradually diminishes in intensity with increasing chase periods,and is no longer detected in the cells by 180 minutes of chase. A 32 kDpolypeptide band also is observed in VEGF-C transfected cells (and notmock-transfected cells). This 32 kD band disappears with similarkinetics to that of the 55 kD band. Simultaneously, increasing amountsof 32 kD (arrow) and subsequently 23 kD (arrow) and 14 kD polypeptidesappear in the medium.

Collectively, the data from the pulse-chase experiments indicate thatthe 55 kD intracellular polypeptide represents a pro-VEGF-C polypeptide,which is not secreted from cells, but rather is first proteolyticallycleaved into the 32 kD form. The 32 kD form is secreted-andsimultaneously further processed by proteolysis into the 23 kD and 14 kDforms. Without intending to be limited to a particular theory, it isbelieved that processing of the VEGF-C precursor occurs as removal of asignal sequence, removal of the COOH-terminal domain (BR3P), and removalof an amino terminal peptide, resulting in a VEGF-C polypeptide havingthe TEE . . . amino terminus.

At high resolution, the 23 kD polypeptide band appears as a closelyspaced polypeptide doublet, suggesting heterogeneity in cleavage orglycosylation.

EXAMPLE 20 Isolation of Mouse cDNA Clones Encoding VEGF-C

To clone a mouse variant of VEGF-C, approximately 1×10⁶ bacteriophagelambda clones of a commercially-available 12 day mouse embryonal cDNAlibrary (lambda EXlox library, Novagen, catalog number 69632-1) werescreened with a radiolabeled fragment of human VEGF-C cDNA containingnucleotides 495 to 1661 of SEQ ID NO: 32. One positive clone wasisolated.

A 1323 bp EcoRI/HindIII fragment of the insert of the isolated mousecDNA clone was subcloned into the corresponding sites of the pBluescriptSK+ vector (Stratagene) and sequenced. The cDNA sequence of this clonewas homologous to the human VEGF-C sequence reported herein, except thatabout 710 bp of 5′-end sequence present in the human clone was notpresent in the mouse clone.

For further screening of mouse cDNA libraries, a HindIII-BstXI (HindIIIsite is from the pBluescript SK+ polylinker) fragment of 881 bp from thecoding region of the mouse cDNA clone was radiolabeled and used as aprobe to screen two additional mouse cDNA libraries. Two additional cDNAclones from an adult mouse heart ZAP II cDNA library (Stratagene,catalog number 936306) were identified. Three additional clones alsowere isolated from a mouse heart 5′-stretch-plus cDNA library in λgt11(Clontech Laboratories, Inc., catalog number ML5002b). Of the latterthree clones, one was found to contain an insert of about 1.9 kb. Theinsert of this cDNA clone was subcloned into EcoRI sites of pBluescriptSK+ vector and both strands of this clone were completely sequenced,resulting in the nucleotide and deduced amino acid sequences shown inSEQ ID NOs: 40 and 41.

It is contemplated that the polypeptide corresponding to SEQ ID NO: 41is processed into a mature mouse VEGF-C protein, in a manner analogousto the processing of the human VEGF-C prepropeptide. Putative cleavagesites for the mouse protein are identified using procedures outlinedabove for identification of cleavage sites for the human VEGF-Cpolypeptide.

The foregoing example demonstrates the utility of polynucleotides of theinvention for-identifying and isolating polynucleotides encoding othernon-human mammalian variants of VEGF-C. Such identified and isolatedpolynucleotides, in turn, can be expressed (using procedures similar tothose described in preceding examples) to produce recombinantpolypeptides corresponding to non-human mammalian variants of VEGF-C.

EXAMPLE 21 N-terminal Peptide Sequence Analyses of Recombinant VEGF-C

Cells (293 EBNA) transfected with VEGF-C cDNA (see Example 13) secreteseveral forms of recombinant VEGF-C (FIG. 21A, lane IP). In the absenceof alkylation, the three major, proteolytically-processed forms ofVEGF-C migrate in SDS-PAGE as proteins with apparent molecular masses of32/29 kD (doublet), 21 kD and 15 kD. Two minor polypeptides exhibitapproximate molecular masses of 63 and 52 kD, respectively. One of thesepolypeptides is presumably a glycosylated and non-processed form; theother polypeptide is presumably glycosylated and partially processed.

To determine sites of proteolytic cleavage of the VEGF-C precursor, animmunoaffinity column was used to purify VEGF-C polypeptides from theconditioned medium of 293 EBNA cells transfected with VEGF-C cDNA. Toprepare the immunoaffinity column, a rabbit was immunized with asynthetic peptide corresponding to the amino-terminus of mature VEGF-Csecreted from the PC-3 cell line (amino acids 104-120 in SEQ ID NO: 33:H₂N-EETIKFAAAHYNTEILK; see PAM126 in Example 19). The IgG fraction wasisolated from the serum of the immunized rabbit using protein ASepharose (Pharmacia). The isolated IgG fraction was covalently bound toCNBr-activated Sepharose CL-4B (Pharmacia) using standard techniques ata concentration of 5 mg IgG/ml of Sepharose. This immunoaffinity matrixwas used to isolate processed VEGF-C from 1.2 liters of the conditionedmedium (CM).

The purified material eluted from the column was analyzed by gelelectrophoresis and Western blotting. Fractions containing VEGF-Cpolypeptides were combined, dialyzed against 10 mM Tris HCl,vacuum-dried, electrotransferred to Immobilon-P (polyvinylidenedifluoride or PVDF) transfer membrane (Millipore, Marlborough, Mass.)and subjected to N-terminal amino acid sequence analysis.

The polypeptide band of 32 kD yielded two distinct sequences:NH₂-FESGLDLSDA . . . and NH₂-AVVMTQTPAS . . . (SEQ ID NO: 51), theformer corresponding to the N-terminal part of VEGF-C after cleavage ofthe signal peptide, starting from amino acid 32 (SEQ ID NO: 33), and thelatter corresponding to the kappa-chain of IgG, which was present in thepurified material due to “leakage” of the affinity matrix during theelution procedure.

In order to obtain the N-terminal peptide sequence of the 29 kD form ofVEGF-C, a construct (VEGF-C NHis) encoding a VEGF-C variant wasgenerated. In particular, the construct encoded a VEGF-C variant thatfused a 6×His tag to the N-terminus of the secreted precursor (i.e.,between amino acids 31 and 33 in SEQ ID NO: 33). The phenylalanine atposition 32 was removed to prevent possible cleavage of the tag sequenceduring secretion of VEGF-C. The VEGF-C NHis construct was cloned intopREP7 as a vector; the construction is described more fully in Example28, below.

The calcium phosphate co-precipitation technique was used to transfectVEGF-C NHis into 293 EBNA cells. Cells were incubated in DMEM/10% fetalcalf serum in 15 cm cell culture dishes (a total of 25 plates). Thefollowing day, the cells were reseeded into fresh culture dishes (75plates) containing the same medium and incubated for 48 hours. Celllayers were then washed once with PBS and DMEM medium lacking FCS wasadded. Cells were incubated in this medium for 48 hours and the mediumwas collected, cleared by centrifugation at 5000×g and concentrated 500×using an Ultrasette Tangential Flow Device (Filtron, Northborough,Mass.), as described in Example 5 above. VEGF-C NHis was purified fromthe concentrated conditioned medium using TALONY™ Metal Affinity Resin(Clontech Laboratories, Inc.) and the manufacturer's protocol for nativeprotein purification using imidazole-containing buffers. The protein waseluted with a solution containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl,and 200 mM imidazole. The eluted fractions containing purified VEGF-CNHis were detected by immunoblotting with Antiserum 882 (antiserum fromrabbit 882). Fractions containing VEGF-C NHis were combined, dialyzedand vacuum-dried. As can be seen in FIG. 27, due to the presence of the6×His tag at the N-terminus of this form of VEGF-C, the upper componentof the major doublet of the VEGF-CNHis migrates slightly slower than the32 kD form of wild type VEGF-C, thereby improving the separation of theVEGF-CNHis 32 kD variant from the 29 kD band using SDS-PAGE.Approximately 15 μg of the purified VEGF-C were subjected to SDS-PAGEunder reducing conditions, electrotransferred to Immobilon-P (PVDF)transfer membrane (Millipore, Inc., Marlborough, Mass.) and the band at29 kD was subjected to N-terminal amino acid sequence analysis. Thissequence analysis revealed an N-terminal sequence of H₂N-SLPAT . . . ,corresponding to amino acids 228-232 of VEGF-C (SEQ ID NO: 33).

The polypeptide band of 21 kD yielded the sequence H₂N-AHYNTEILKS . . ., corresponding to an amino-terminus starting at amino acid 112 of SEQID NO: 33. Thus, the proteolytic processing site which results in the 21kD form of VEGF-C produced by transfected 293 EBNA cells apparentlyoccurs nine amino acid residues downstream of the cleavage site whichresults in the 23 kD form of VEGF-C secreted by PC-3 cells.

The N-terminus of the 15 kD form was identical to the N-terminus of the32 kD form (NH₂-FESGLDLSDA . . .). The 15 kD form was not detected whenrecombinant VEGF-C was produced by COS cells. This suggests thatproduction of this form is cell lineage specific.

EXAMPLE 22 Dimeric and Monomeric Forms of VEGF-C

The composition of VEGF-C dimers was analyzed as follows. Cells (293EBNA cells), transfected with the pREP7 VEGF-C vector as described inExample 11, were metabolically labelled with Pro-mix L-[³⁵S] labellingmix (Amersham Corp.) to a final concentration of 100 μCi/ml.

In parallel, a VEGF-C mutant, designated “R102S”, was prepared andanalyzed. To prepare the DNA encoding VEGF-C-R102S, the arginine codonat position 102 of SEQ ID NO: 33 was replaced with a serine codon. ThisVEGF-C-R102S-encoding DNA, in a pREP7 vector, was transfected into 293EBNA cells and expressed as described above. VEGF-C polypeptides wereimmunoprecipitated using antisera 882 (obtained by immunization of arabbit with a polypeptide corresponding to residues 104-120 of SEQ IDNO: 33 (see previous Example)) and antisera 905 (obtained byimmunization of a rabbit with a polypeptide corresponding to a portionof the prepro-VEGF-C leader: H₂N-ESGLDLSDAEPDAGEATAYASK (residues 33 to54 of SEQ ID NO: 33).

The immunoprecipitates from each cell culture were subjected to SDS-PAGEunder non-denaturing conditions (FIG. 21B). Bands 1-6 were cut out fromthe gel, soaked for 30 minutes in 1×gel-loading buffer containing 200 mMβ-mercaptoethanol, and individually subjected to SDS-PAGE underdenaturing conditions (FIGS. 21A and 21C, lanes 1-6).

As can be seen from FIGS. 21A-C, each high molecular weight form ofVEGF-C (FIG. 21B, bands 1-4) consists of at least two monomers bound bydisulfide bonds (Compare FIGS. 21A and 21C, lanes 1-4, in the reducinggels). The main component of bands 1-3 is the doublet of 32/29 kD, whereboth proteins are present in an equimolar ratio. The main fraction ofthe 21 kD form is secreted as either a monomer or as a homodimerconnected by means other than disulfide bonds (bands 6 and lanes 6 inFIGS. 21A-C).

The R102S mutation creates an additional site for N-linked glycosylationin VEGF-C at the asparagine residue at position 100 in SEQ ID NO: 33.Glycosylation at this additional glycosylation site increases theapparent molecular weight of peptides containing the site, as confirmedin FIGS. 21A-C and FIGS. 22A-B. The additional glycosylation lowers themobility of forms of VEGF-C-R102S that contain the additionalglycosylation site, when compared to peptides of similar primarystructure corresponding to VEGF-C. FIGS. 21A-C and FIGS. 22A-B revealthat the VEGF-C-R102S polypeptides corresponding to the 32 kD and 15 kDforms of wt VEGF-C exhibit increased apparent molecular weights,indicating that each of these peptides contains the newly introducedglycosylation site. In particular, the VEGF-C-R102S peptidecorresponding to the 15 kD peptide from VEGF-C comigrates on a gel withthe 21 kD form of the wild type (wt) VEGF-C, reflecting a shift on thegel to a position corresponding to a greater apparent molecular weight.(Compare lanes 4 in FIGS. 21A and 21C).

In a related experiment, another VEGF-C mutant, designated “R226,227S,”was prepared and analyzed. To prepare a DNA encoding VEGF-C-R226,227S,the arginine codons at positions 226 and 227 of SEQ ID NO: 33 werereplaced with serine codons by site-directed mutagenesis. The resultantDNA was transfected into 293 EBNA cells as described above and expressedand analyzed under the same conditions as described for VEGF-C andVEGF-C-R102S. In the conditioned medium from the cells expressingVEGF-C-R226,227S, no 32 kD form of VEGF-C was detected. These resultsindicate that a C-terminal cleavage site of wild-type VEGF-C is adjacentto residues 226 and 227 of SEQ ID NO: 33, and is destroyed by themutation of the arginines to serines. Again, the mobility of the 29 kDcomponent of the doublet was unchanged (FIGS. 22A-B).

Taken together, these data indicate that the major form of the processedVEGF-C is a heterodimer consisting of (1) a polypeptide of 32 kDcontaining amino acids 32-227 of the prepro-VEGF-C (amino acids 32 to227 in SEQ ID NO: 33) attached by disulfide bonds to (2) a polypeptideof 29 kD beginning with amino acid 228 in SEQ ID NO: 33. These data arealso supported by a comparison of the pattern of immunoprecipitated,labelled VEGF-C forms using antisera 882 and antisera 905.

When VEGF-C immunoprecipitation was carried out using conditionedmedium, both antisera (882 and 905) recognized some or all of the threemajor processed forms of VEGF-C (32/29 kD, 21 kD and 15 kD). When theconditioned medium was reduced by incubation in the presence of 10 mMdithiothreitol for two hours at room temperature with subsequentalkylation by additional incubation with 25 mM iodoacetamide for 20minutes at room temperature, neither antibody precipitated the 29 kDcomponent, although antibody 882 still recognized polypeptides of 32 kD,21 kD and 15 kD. These results are consistent with the nature of thepeptide antigen used to elicit the antibodies contained in antisera 882,a peptide containing amino acids 2-18 of SEQ ID NO: 33. On the otherhand, antisera 905 recognized only the 32 kD and 15 kD polypeptides,which include sequence of the peptide (amino acids 33 to 54 of SEQ IDNO: 33) used for immunization to obtain antisera 905. Taking intoaccount the mobility shift of the 32 kD and 15 kD forms, theimmunoprecipitation results with the R102S mutant were similar (FIGS.23A-B). The specificity of antibody 905 is confirmed by the fact that itdid not recognize a VEGF-C ΔN variant form wherein the N-terminalpropeptide spanning residues 32-102 of the unprocessed polypeptide hadbeen deleted (FIG. 23B).

The results of these experiments also demonstrate that the 21 kDpolypeptide is found (1) in heterodimers with other molecular forms (seeFIGS. 21A-C and FIGS. 22A-B), and (2) secreted as a monomer or ahomodimer held by bonds other than disulfide bonds (FIGS. 21A and 21B,lanes 6).

The experiments disclosed in this example demonstrate that several formsof VEGF-C exist. A variety of VEGF-C monomers were observed and thesemonomers can vary depending on the level and pattern of glycosylation.In addition, VEGF-C was observed as a multimer, for example a homodimeror a heterodimer. The processing of VEGF-C is schematically presented inFIG. 37 (disulfide bonds not shown). All forms of VEGF-C are within thescope of the present invention.

EXAMPLE 23 In Situ Hybridization of Mouse Embryos

To analyze VEGF-C mRNA distribution in different cells and tissues,sections of 12.5 and 14.5-day post-coitus (p.c.) mouse embryos wereprepared and analyzed via in situ hybridization using labeled VEGF-Cprobes. In situ hybridization of tissue sections was performed asdescribed Västrik et al., J. Cell Biol., 128:1197-1208 (1995). A mouseVEGF-C antisense RNA probe was generated from linearized pBluescript IISK+ plasmid (Stratagene Inc., La Jolla, Calif.), containing a cDNAfragment corresponding to nucleotides 499-979 of a mouse VEGF-C cDNA(SEQ ID NO: 40). This clone was obtained from a partial clone isolatedfrom the Novagen library (see the 1322 bp cDNA clone of FIG. 31A), fromwhich the 3′ noncoding region and the BR3P repeats had been removed byexonuclease III treatment. Radiolabeled RNA was synthesized using T7polymerase and [³⁵S]-UTP (Amersham). Mouse VEGF-B antisense and senseRNA probes were synthesized in a similar manner from linearized pCRIIplasmid containing the mouse VEGF-B cDNA insert as described Olofsson etal., Proc. Natl. Acad. Sci. (USA), 93:2576-2581 (1996). The highstringency wash was for 45 minutes at 65° C. in a solution containing 30mM DTT and 4×SSC. The slides were exposed for 28 days, developed andstained with hematoxylin. For comparison, similar sections werehybridized with a VEGFR-3 probe and the 12.5-day p.c. embryos were alsoprobed for VEGF-B mRNA.

FIGS. 34A-D show darkfield (FIGS. 34A-C) and lightfield (FIG. 34D)photomicrographs of 12.5 day p.c. embryo sections probed with theantisense (FIG. 34A) and sense (FIGS. 34C-D) VEGF-C probes. FIG. 34Aillustrates a parasagittal section, where VEGF-C mRNA is particularlyprominent in the mesenchyme around the vessels surrounding thedeveloping metanephros (mn). In addition, hybridization signals wereobserved between the developing vertebrae (vc), in the developing lungmesenchyme (lu), in the neck region and developing forehead. Thespecificity of these signals is evident from the comparison with VEGF-Bexpression in an adjacent section (FIG. 34B), where the myocardium givesa very strong signal and lower levels of VEGF-B mRNA are detected inseveral other tissues. Both genes appear to be expressed in between thedeveloping vertebrae (vc), in the developing lung (lu) and forehead.Hybridization of the VEGF-C sense probe showed no specific expressionwithin these structures (FIG. 34C).

FIGS. 35A-D show a comparison of the expression patterns of VEGF-C andVEGFR-3 in 12.5 day p.c. mouse embryos in the jugular region, where thedeveloping dorsal aorta and cardinal vein are located. This is the areawhere the first lymphatic vessels sprout from venous sac-like structuresaccording to a long-standing theory. Sabin, Am. J. Anat., 9:43-91(1909). As can be seen from FIGS. 35A-D, an intense VEGF-C signal isdetected in the mesenchyme surrounding the developing venous sacs (FIGS.35A and 35C) which are positive for VEGFR-3 (FIGS. 35B and 35D).

The mesenterium supplies the developing gut with blood and containsdeveloping lymphatic vessels. The developing 14.5 day p.c. mesenteriumis positive for VEGF-C mRNA, with particularly high expression inconnective tissue surrounding certain vessels (arrowheads in FIGS.35A-H). This signal in FIG. 35E should be distinguished from the falsepositive reflection of light from red blood cells within the vessel,shown with an asterisk. The adjacent mesenterial VEGFR-3 signals shownin FIG. 35F originate from small capillaries of the mesenterium(arrowheads). Therefore, there appears to be a paracrine relationshipbetween the production of the mRNAs for the two growth factors. Thisdata indicates that VEGF-C is expressed in a variety of tissues.Moreover, the pattern of expression is consistent with a role for VEGF-Cin venous and lymphatic vessel development. Further, the data revealsthat VEGF-C is expressed in non-human animals.

EXAMPLE 24 Analysis of VEGF, VEGF-B, and VEGF-C mRNA Expression in Fetaland Adult Tissues

A human fetal tissue Northern blot containing 2 μg of polyadenylatedRNAs from brain, lung, liver and kidney (Clontech Inc.) was hybridizedwith a pool of the following probes: a human full-length VEGF-C cDNAinsert (Genbank Acc. No. X94216), a human VEGF-B,₆₇ cDNA fragment(nucleotides 1-382, Genbank Acc. No. U48800) obtained by PCRamplification; and a human VEGF 581 bp cDNA fragment covering base pairs57-638 (Genbank Acc. No. X15997). Blots were washed under stringentconditions, using techniques standard in the art.

Mouse embryo multiple tissue Northern blot (Clontech Inc.) containing 2μg of polyadenylated RNAs from 7, 11, 15 and 17 day postcoital (p.c.)embryos was hybridized with mouse VEGF-C cDNA fragment (base pairs499-656). A mouse adult tissue Northern blot was hybridized with theprobes for human VEGF, VEGF-B,₆₇, VEGF-C and with a VEGFR-3 cDNAfragment (nucleotides 1-595; Genbank Acc. No. X68203).

In adult mouse tissues, both 2.4 kb and 2.0 kb mRNA signals wereobserved with the VEGF-C probe, at an approximately 4:1 ratio. The mostconspicuous signals were obtained from lung and heart RNA, while kidney,liver, brain, and skeletal muscle had lower levels, and spleen andtestis had barely visible levels (FIG. 33A). As in the human tissues,VEGF mRNA expression in adult mice was most abundant in lung and heartRNA, whereas the other samples showed less coordinate regulation withVEGF-C expression. Skeletal muscle and heart tissues gave the highestVEGF-B mRNA levels from adult mice, as previously reported Olofsson etal., Proc. Natl. Acad. Sci. (USA), 93:2576-2581 (1996). Comparison withVEGFR-3 expression showed that the tissues where VEGF-C is expressedalso contain mRNA for its cognate receptor tyrosine kinase, although inthe adult liver VEGFR-3 mRNA was disproportionally abundant.

To provide a better insight into the regulation of the VEGF-C mRNAduring embryonic development, polyadenylated RNA isolated from mouseembryos of various gestational ages (7-17 day p.c.) was hybridized withthe mouse VEGF-C probe. These analyses showed that the amount of 2.4 kbVEGF-C mRNA is relatively constant throughout the gestational period(FIG. 33B).

EXAMPLE 25 Regulation of mRNAs for VEGF Family Members by Serum,Interleukin-1 and Dexamethasone in Human Fibroblasts in Culture

Human IMR-90 fibroblasts were grown in DMEM medium containing 10% FCSand antibiotics. The cells were grown to 80% confluence, then starvedfor 48 hours in 0.5% FCS in DMEM. Thereafter, the growth medium waschanged to DMEM containing 5% FCS, with or without 10 ng/mlInterleukin-1 (IL-1) and with or without 1 mM dexamethasone, asindicated in FIGS. 25A-B. The culture plates were incubated with theseadditions for the times indicated, and total cellular RNA was isolatedusing the TRIZOL kit (GIBCO-BRL). About 20 μg of total RNA from eachsample was electrophoresed in 1.5% formaldehyde-agarose gels asdescribed in Sambrook et al., supra (1989). The gel was used forNorthern blotting and hybridization with radiolabeled insert DNA fromthe human VEGF (VEGF-A) clone (a 581 bp cDNA covering bps 57-638,Genbank Acc. No. 15997) and a human VEGF-B₁₆₇ cDNA fragment (nucleotides1-382, Genbank Acc. No. U48800) (FIG. 25B). Subsequently, the Northernblots were probed with radiolabelled insert from the VEGF-C cDNA plasmid(FIG. 25A). Primers were labelled using a standard technique involvingenzymatic extension reactions of random primers, as would be understoodby one of ordinary skill in the art. The mobilities of the 28S and 18Sribosomal RNA bands are indicated, based on UV photography of ethidiumbromide stained RNA before the transfer.

As can be seen in FIGS. 25A-B, very low levels of VEGF-C and VEGF-A areexpressed by the starved IMR-90 cells as well as cells after 1 hour ofstimulation. In contrast, abundant VEGF-B mRNA signal is visible underthese conditions. After a 4 hours of serum stimulation, there is astrong induction of VEGF-C and VEGF mRNAs, which are further increasedin the IL-1 treated sample. The effect of IL-1 seems to be abolished inthe presence of dexamethasone. A similar pattern of enhancement ismaintained in the 8 hour sample, but a gradual down-regulation of allsignals occurs for both RNAs in the 24 hour and 48 hour samples. Incontrast, VEGF-B mRNA levels remain constant and thus show remarkablestability throughout the time period. The results are useful in guidingefforts to use VEGF-C in methods for treating a variety of disorders.

EXAMPLE 26 Expression and Analysis of Recombinant VEGF-C

The mouse VEGF-C cDNA was expressed as a recombinant protein and thesecreted protein was analyzed for its receptor binding properties. Thebinding of mouse VEGF-C to the human VEGFR-3 extracellular domain wasstudied by using media from Bosc23 cells transfected with mouse VEGF-CcDNA in a retroviral expression vector.

The 1.8 kb mouse VEGF-C cDNA was cloned as an EcoRI fragment into theretroviral expression vector pBabe-puro containing the SV40 earlypromoter region Morgenstern et al., Nucl. Acids Res., 18:3587-3595(1990), and transfected into the Bosc23 packaging cell line by thecalcium-phosphate precipitation method Pearet et al., Proc. Natl. Acad.Sci. (USA), 90:8392-8396 (1994). For comparison, Bosc23 cells also weretransfected with the previously-described human VEGF-C construct in thepREP7 expression vector. The transfected cells were cultured for 48hours prior to metabolic labelling. Cells were changed into DMEM mediumdevoid of cysteine and methionine, and, after 45 minutes ofpreincubation and medium change, Pro-mix™ L-[³⁵] in vitro cell labellingmix (Amersham Corp.), in the same medium, was added to a finalconcentration of about 120 μCi/ml. After 6 hours of incubation, theculture medium was collected and clarified by centrifugation.

For immunoprecipitation, 1 ml aliquots of the media frommetabolically-labelled Bosc23 cells transfected with empty vector ormouse or human recombinant VEGF-C, respectively, were incubatedovernight on ice with 2 μl of rabbit polyclonal antiserum raised againstan N-terminal 17 amino acid peptide of mature human VEGF-C(H₂N-EETIKFAAAHYNTEILK) (SEQ ID NO: 33, residues 104-120). Thereafter,the samples were incubated with protein A sepharose for 40 minutes at 4°C. with gentle agitation. The sepharose beads were then washed twicewith immunoprecipitation buffer and four times with 20 mM Tris-HCl, pH7.4. Samples were boiled in Laemmli buffer and analyzed by 12.5% sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Immunoprecipitation of VEGF-C from media of transfected andmetabolically-labelled cells revealed bands of approximately 30-32×10³M_(r) (a doublet) and 22-23×10³ M_(r) in 12.5% SDS-PAGE. These bandswere not detected in samples from nontransfected or mock-transfectedcells as shown in FIG. 32A (i.e., lanes marked “vector”). These resultsshow that antibodies raised against human VEGF-C recognize thecorresponding mouse ligand.

For receptor binding experiments, 1 ml aliquots of media frommetabolically-labelled Bosc23 cells were incubated with VEGFR-3extracellular domain covalently coupled to sepharose for 4 hours at 4°C. with gentle mixing. The VEGFR-3 extracellular domain is described inco-owned, co-pending U.S. patent application Ser. No. 08/340,011,incorporated herein by reference. The sepharose beads were washed fourtimes with ice-cold phosphate buffered saline (PBS), and the sampleswere analyzed by gel electrophoresis as described in Joukov et al., EMBOJ., 15:290-298 (1996).

As can be seen from FIG. 32A, similar 30-32×10³ M_(r) doublet and22-23×10³ M_(r) polypeptide bands were obtained in the receptor bindingassay as compared to the immunoprecipitation assay. Thus, mouse VEGF-Cbinds to human VEGFR-3. The slightly faster mobility of the mouse VEGF-Cpolypeptides may be caused by the four amino acid residue differenceobserved in sequence analysis (residues H88-E91, FIGS. 32B and 32C).

The capacity of mouse recombinant VEGF-C to induce VEGFR-3autophosphorylation was also investigated. For the VEGFR-3 receptorstimulation experiments, subconfluent NIH 3T3-VEGFR-3 cells, Pajusola etal., Oncogene, 9:3545-3555 (1994), were starved overnight in serum-freemedium containing 0.2% BSA. In general, the cells were stimulated withthe conditioned medium from VEGF-C vector-transfected cells for 5minutes, washed three times with cold PBS containing 200 μM vanadate,and lysed in RIPA buffer for immunoprecipitation analysis. The lysateswere centrifuged for 25 minutes at 16000×g and the resultingsupernatants were incubated for 2 hours on ice with the specificantisera, followed by immunoprecipitation using protein A-sepharose andanalysis in 7% SDS-PAGE. Polypeptides were transferred to nitrocelluloseand analyzed by immunoblotting using anti-phosphotyrosine (TransductionLaboratories) and anti-receptor antibodies, as described by Pajusola etal., Oncogene, 9:3545-3555 (1994). Filter stripping was carried out at50° C. for 30 minutes in 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mMTris-HCl, pH 6.7, with occasional agitation. U.S. patent applicationSer. No. 08/340,011. The results of the experiment are shown in FIGS.32B and 32C. The results demonstrate that culture medium containingmouse VEGF-C stimulates the autophosphorylation of VEGFR-3 to a similarextent as human baculoviral VEGF-C or the tyrosyl phosphatase inhibitorpervanadate.

VEGFR-2 stimulation was studied in subconfluent porcine aorticendothelial (PAE) cells expressing VEGFR-2 (PAE-VEGFR-2) Waltenberger etal., J. Biol. Chem., 269:26988-26995 (1994), which were starvedovernight in serum-free medium containing 0.2% BSA. Stimulation wascarried out and the lysates prepared as described above. For receptorimmunoprecipitation, specific antiserum for VEGFR-2 (Waltenberger etal., J. Biol. Chem., 269:26988-26995 (1994) was used. Theimmunoprecipitates were analyzed as described for VEGFR-3 in 7% SDS-PAGEfollowed by Western blotting with anti-phosphotyrosine antibodies,stripping of the filter, and re-probing it with anti-VEGFR-2 antibodies(Santa Cruz).

Mouse VEGF-C appeared to be a potent inducer of VEGFR-3autophosphorylation, with the 195×10³ M_(r) precursor andproteolytically cleaved 125×103 M_(r) tyrosine kinase polypeptides ofthe receptor (Pajusola et al., Oncogene, 9.3545-3555 (1994)), beingphosphorylated. VEGFR-2 stimulation was first tried with unconcentratedmedium from cells expressing recombinant VEGF-C, but immunoblottinganalysis did not reveal any receptor autophosphorylation.

To further determine whether mouse recombinant VEGF-C can also induceVEGFR-2 autophosphorylation as has been previously reported for humanVEGF-C (Joukov et al., EMBO J., 15:290-298 (1996)), PAE cells expressingVEGFR-2 were stimulated with tenfold concentrated medium from culturestransfected with mouse VEGF-C expression vector and autophosphorylationwas analyzed. For comparison, cells treated with tenfold concentratedmedium containing human recombinant VEGF-C (Joukov et al., (1996)),unconcentrated medium from human VEGF-C baculovirus infected insectcells, or pervanadate (a tyrosyl phosphatase inhibitor) were used. Ascan be seen from FIGS. 32B and 32C, in response to human baculoviralVEGF-C as well as pervanadate treatment, VEGFR-2 was prominentlyphosphorylated, whereas human and mouse recombinant VEGF-C gave a weakand barely detectable enhancement of autophosphorylation, respectively.Media from cell cultures transfected with empty vector or VEGF-C clonedin the antisense orientation did not induce autophosphorylation ofVEGFR-2. Therefore, mouse VEGF-C binds to VEGFR-3 and activates thisreceptor at a much lower concentration than needed for the activation ofVEGFR-2. Nevertheless, the invention comprehends methods for using thematerials of the invention to take advantage of the interaction ofVEGF-C with VEGFR-2, in addition to the interaction between VEGF-C andVEGFR-3.

EXAMPLE 27 VEGF-C T103-S213 Fragment Expressed in Pichia YeastStimulates Autophosphorylation of Flt4 (VEGFR-3) and KDR (VEGFR-2)

A truncated form of human VEGF-C cDNA was constructed wherein (1) thesequence encoding residues of the putative mature VEGF-C amino terminusH₂N-E(104)ETIK(SEQ ID NO: 33, residues 104 et seq.) was fused to theyeast PHO1 signal sequence, and (2) a stop codon was introduced afteramino acid 213 (H₂N- . . . RCMS; i.e., after codon 213 of SEQ ID NO:32). The resultant truncated cDNA construct was then inserted into thePichia pastoris expression vector pHIL-S1. The engineering of thisconstruct is schematically illustrated in FIG. 25. For the cloning, aninternal BglII site in the VEGF-C coding sequence was mutated withoutchange of the encoded polypeptide sequence.

This VEGF-C expression vector was then transfected into Pichia cells andpositive clones were identified by screening for the expression ofVEGF-C protein in the culture medium by Western blotting. One positiveclone was grown in a 50 ml culture, and induced with methanol forvarious periods of time from 0 to 60 hours. About 10 μl of medium wasanalyzed by gel electrophoresis, followed by Western blotting anddetection with anti-VEGF-C antiserum, as described above. As can be seenin FIG. 26A, an approximately 24 kD polypeptide (note the band spreadingdue to glycosylation) accumulates in the culture medium of cellstransfected with the recombinant VEGF-C construct, but not in the mediumof mock-transfected cells or cells transfected with the vector alone.

The medium containing the recombinant VEGF-C protein was concentrated byCentricon 30 kD cutoff ultrafiltration and used to stimulate NIH 3T3expressing Flt4 (VEGFR-3) and porcine aortic endothelial (PAE) cellsexpressing KDR (VEGFR-2). The stimulated cells were lysed andimmunoprecipitated using VEGFR-specific antisera and theimmunoprecipitates were analyzed by Western blotting usinganti-phosphotyrosine antibodies, chemiluminescence, and fluorography. Asa positive control for maximal autophosphorylation of the VEGFRs,vanadate (VO₄) treatment of the cells for 10 min was used. As can beseen from the results shown in FIG. 26B, medium from Pichia culturessecreting this recombinant VEGF-C polypeptide inducesautophosphorylation of both Flt4l polypeptides of 195 kD and 125 kD aswell as the KDR polypeptide of about 200 kD. Vanadate, on the otherhand, induces heavy tyrosyl phosphorylation of the receptor bands inaddition to other bands probably coprecipitating with the receptors.

These results demonstrate that a VEGF-homologous domain of VEGF-Cconsisting of amino acid residues 104E -213S (SEQ ID NO: 33, residues104-213) can be recombinantly produced in yeast and is capable ofstimulating the autophosphorylation of Flt4 (VEGFR-3) and KDR (VEGFR-2).Recombinant VEGF-C fragments such as the fragment described herein,which are capable of stimulating Flt4 or KDR autophosphorylation areintended as aspects of the invention; methods of using these fragmentsare also within the scope of the invention.

EXAMPLE 28 Properties of the Differentially Processed Forms of VEGF-C

The following oligonucleotides were used to generate a set of VEGF-Cvariants:

5′-TCTCTTCTGTGCTTGAGTTGAG-3′ (SEQ ID NO: 42), used to generate VEGF-CR102S (arginine mutated to serine at position 102 (SEQ ID NO: 33));

5′-TCTCTTCTGTCCCTGAGTTGAG-3′ (SEQ ID NO: 43), used to generate VEGF-CR102G (arginine mutated to glycine at position 102 (SEQ ID NO: 33));

5′-TGTGCTGCAGCAAATTTTATAGTCTCTTCTGTGGCGGCGGC GGCGGCGGGCGCCTCGCGAGGACC-3′(SEQ ID NO: 44), used to generate VEGF-C ΔN (deletion of N-terminalpropeptide corresponding to amino acids 32-102 (SEQ ID NO: 33));

5′-CTGGCAGGGAACTGCTAATAATGGAATGAA-3′ (SEQ ID NO: 45), used to generateVEGF-C R226,227S (arginine codons mutated to serines at positions 226and 227 (SEQ ID NO: 33));

5 ′-GGGCTCCGCGTCCGAGAGGTCGAGTCCGGACTCGTGATGGTGATGGTGATGGGCGGCGGCGGCGGCGGGCGCCTCGCGAGGACC-3′ (SEQ ID NO: 46), used togenerate VEGF-C NHis (this construct encodes a polypeptide with a 6×Histag fused to the N-terminus of the secreted precursor (amino acid 32 ofSEQ ID NO: 33)).

Some of the foregoing VEGF-C variant constructs were further modified toobtain additional constructs. For example, VEGF-C R102G in pALTER andoligonucleotide 5′-GTATTATAATGTCCTCCACCAAATTTTATAG-3′ (SEQ ID NO: 47)were used to generate VEGF-C 4G, which encodes a polypeptide with fourpoint mutations: R102G, A110G, A111G, and A112G (alanines mutated toglycines at positions 110-112 (SEQ ID NO: 33). These four mutations areadjacent to predicted sites of cleavage of VEGF-C expressed in PC-3 andrecombinantly expressed in 293 EBNA cells.

Another construct was created using VEGF-C ΔN and oligonucleotide5′-GTTCGCTGCCTGACACTGTGGTAGTGTTGCTGGCGGCCGCTAGTGATGGTGATGGTGATGAATAATGGAATGAACTTGTCTGTAAACATCCAG-3′(SEQ ID NO: 48) to generate VEGF-C ΔNΔCHis. This construct encodes apolypeptide with a deleted N-terminal propeptide (amino acids 32-102); adeleted C-terminal propeptide (amino acids 226-419 of SEQ ID NO: 33);and an added 6×His tag at the C-terminus.

All constructs were further digested with HindIII and NotI, subclonedinto HindIII/NotI digested pREP7 vector, and used to transfect 293 EBNAcells. About 48 hours after transfection, the cells were eithermetabolically labelled with ³⁵S as described above, or starved inserum-free medium for 2 days. Media were then collected and used insubsequent experiments. As can be seen from FIGS. 27A-B, wild type (wt)VEGF-C, VEGF-C NHis and VEGF-C ΔNΔCHis were expressed to similar levelsin 293 EBNA cells. At the same time, expression of the VEGF-C 4Gpolypeptide was considerably lower, possibly due to the changedconformation and decreased stability of the translated product. However,all the above VEGF-C variants were secreted from the cells (compareFIGS. 27A and 27B). The conditioned media from the transfected andstarved cells were concentrated 5-fold and used to assess their abilityto stimulate tyrosine phosphorylation of Flt4 (VEGFR-3) expressed in NIH3T3 cells and KDR (VEGFR-2) expressed in PAE cells.

FIGS. 28A-B show that wt VEGF-C, as well as all three mutantpolypeptides, stimulate tyrosine phosphorylation of VEGFR-3. The mostprominent stimulation is by the short mature VEGF-C ΔNΔCHis. Thismutant, as well as VEGF-C NHis, also stimulated tyrosine phosphorylationof VEGFR-2. Thus, despite the fact that a major component of secretedrecombinant VEGF-C is a dimer of 32/29 kD, the active part of VEGF-Cresponsible for its binding to VEGFR-3 and VEGFR-2 is localized betweenamino acids 102 and 226 (SEQ ID NO: 33) of the VEGF-C precursor.Analysis and comparison of binding properties and biological activitiesof these VEGF-C proteins and variants, using assays such as thosedescribed herein, will provide data concerning the significance of theobserved major 32/29 kD and 21-23 kD VEGF-C processed forms. The dataindicate that constructs encoding amino acid residues 103-225 of theVEGF-C precursor (SEQ ID NO: 33) generate a recombinant ligand that isfunctional for both VEGFR-3 and VEGFR-2.

The data from this and preceding examples demonstrate that numerousfragments of the VEGF-C polypeptide retain biological activity. Anaturally occurring VEGF-C polypeptide spanning amino acids 103-226 (or103-227) of SEQ ID NO: 33, produced by a natural processing cleavagedefining the C-terminus, has been shown to be active. The foregoingexample demonstrates that a fragment with residues 104-213 of SEQ ID NO:33 retains biological activity.

In addition, data from Example 21 demonstrates that a VEGF-C polypeptidehaving its amino terminus at position 112 of SEQ ID NO: 33 retainsactivity. Additional experiments have shown that a fragment lackingresidues 1-112 of SEQ ID NO: 33 retains biological activity.

In a related experiment, a stop codon was substituted for the lysine atposition 214 of SEQ ID NO: 33 (SEQ ID NO: 32, nucleotides 991-993). Theresulting recombinant polypeptide still was capable of inducing Flt4autophosphorylation, indicating that a peptide spanning amino acidresidues 113-213 of SEQ ID NO: 33 is biologically active.

Sequence comparisons of members of the VEGF family of polypeptidesprovides an indication that still smaller fragments of the polypeptidedepicted in SEQ ID NO: 33 will retain biological activity. Inparticular, eight highly conserved cysteine residues of the VEGF familyof polypeptides define a region from residues 131-211 of SEQ ID NO: 33(see FIGS. 32B and 32C) of evolutionary signficance; therefore, apolypeptide spanning from about residue 131 to about residue 211 isexpected to retain VEGF-C biological activity. In fact, a polypeptidewhich retains the conserved motif RCXXCC (e.g., a polypeptide comprisingfrom about residue 161 to about residue 211 of SEQ ID NO: 33 ispostulated to retain VEGF-C biological activity. To maintain nativeconformation of these fragments, it may be preferred to retain about 1-2additional amino acids at the carboxy-terminus and 1-2 or more aminoacids at the amino terminus.

Beyond the preceding considerations, evidence exists that smallerfragments and/or fragment variants which lack the conserved cysteinesnonetheless will retain VEGF-C biological activity. In particular,VEGF-C that has been reduced and alkylated (processes that interferewith the chemistry of cysteine residues) retains biological activity.Consequently, the materials and methods of the invention include allVEGF-C fragments and variants that retain at least one biologicalactivity of VEGF-C, regardless of the presence or absence of members ofthe conserved set of cysteine residues.

EXAMPLE 29 Expression of Human VEGF-C Under the Human K14 KeratinPromoter in Transgenic Mice Induces Abundant Growth of Lymphatic Vesselsin the Skin

The FLT4 receptor tyrosine kinase is relatively specifically expressedin the endothelia of lymphatic vessels. Kaipainen et al., Proc. Natl.Acad. Sci. (USA), 92: 3566-3570 (1995). Furthermore, the VEGF-C growthfactor stimulates the FLT4 receptor, showing at least 10 to 100 foldless activity towards the KDR receptor of blood vessels (Joukov et al.,EMBO J., 15: 290-298 (1996); Kukk et al., submitted for publication).

Experiments were conducted in transgenic mice to analyze the specificeffects of VEGF-C overexpression in tissues. The human K14 keratinpromoter is active in the basal cells of stratified squamous epithelia(Vassar et al., Proc. Natl. Acad. Sci. (USA), 86:1563-1567 (1989)) andwas used as the expression control element in the recombinant VEGF-Ctransgene. The vector containing the K14 keratin promoter is describedin Vassar et al., Genes Dev., 5:714-727 (1991) and Nelson et al., J.Cell Biol. 97:244-251 (1983).

The recombinant VEGF-C transgene was constructed using the human fulllength VEGF-C cDNA (GenBank Acc. No. X94216). This sequence was excisedfrom a pCI-neo vector (Promega) with XhoI/NotI, and the resulting 2027base pair fragment containing the open reading frame and stop codon(nucleotides 352-1611 of SEQ ID NO: 32) was isolated. The isolatedfragment was then subjected to an end-filling reaction using the Klenowfragment of DNA polymerase. The blunted fragment was then ligated to asimilarly opened BamHI restriction site in the K14 vector. The resultingconstruct contained the EcoRI site derived from the polylinker of thepCI-neo vector. This EcoRI site was removed using standard techniques (aKlenow-mediated fill-in reaction following partial digestion of therecombinant intermediate with EcoRI) to facilitate the subsequentexcision of the DNA fragment to be injected into fertilized mouseoocytes. The resulting clone, designated K14-VEGF-C, is illustrated inFIG. 20.

The EcoRI-HindIII fragment from clone K14 VEGF-C containing the K14promoter, VEGF-C cDNA, and K14 polyadenylation signal was isolated andinjected into fertilized oocytes of the FVB-NIH mouse strain. Theinjected zygotes were transplanted to oviducts of pseudopregnantC57BL/6×DBA/2J hybrid mice. The resulting founder mice were analyzed forthe presence of the transgene by polymerase chain reaction of tail DNAusing the primers: 5′-CATGTACGAACCGCCAG-3′ (SEQ ID NO: 49) and5′-AATGACCAGAGAGAGGCGAG-3′ (SEQ ID NO: 50). In addition, the tail DNAswere subjected to EcoRV digestion and subsequent Southern analysis usingthe EcoRI-HindIII fragment injected into the mice. Out of 8 pupsanalyzed at 3 weeks of age, 2 were positive, having approximately 40-50copies and 4-6 copies of the transgene in their respective genomes.

The mouse with the high copy number transgene was small, developed moreslowly than its litter mates and had difficulty eating (i.e., suckling).Further examination showed a swollen, red snout and poor fur. Althoughfed with a special liquid diet, it suffered from edema of the upperrespiratory and digestive tracts after feeding and had breathingdifficulties. This mouse died eight weeks after birth and wasimmediately processed for histology, immunohistochemistry, and in situhybridization.

Histological examination showed that in comparison to the skin oflittermates, the dorsal dermis of K14-VEGF-C transgenic mice wasatrophic and connective tissue was replaced by large lacunae devoid ofred cells, but lined with a thin endothelial layer (white arrows inFIGS. 29A-D). These distended vessel-like structures resembled thoseseen in human lymphangiomas. The number of skin adnexal organs and hairfollicles were reduced. In the snout region, an increased number ofvessels was also seen. Therefore, VEGF-C overexpression in the basalepidermis is capable of promoting the growth of extensive vesselstructure in the underlying skin, including large vessel lacunae. Thevessel morphology indicates that VEGF-C stimulates the growth of vesselshaving features of lymphatic vessels.

The foregoing in vivo data indicates utilities for both (i) VEGF-Cpolypeptides and polypeptide variants having VEGF-C biological activity,and (ii) anti-VEGF-C antibodies and VEGF-C antagonists that inhibitVEGF-C activity (e.g., by binding VEGF-C or interfering withVEGF-C/receptor interactions. For example, the data indicates atherapeutic utility for VEGF-C polypeptides in patients wherein growthof lymphatic tissue may be desirable (e.g., in patients following breastcancer or other surgery where lymphatic tissue has been removed andwhere lymphatic drainage has therefore been compromised, resulting inswelling; or in patients suffering from elephantiasis). The dataindicates a therapeutic utility for anti-VEGF-C antibody substances andVEGF-C antagonists for conditions wherein growth-inhibition of lymphatictissue may be desirable (e.g., treatment of lymphangiomas). Accordingly,methods of administering VEGF-C and VEGF-C variants and antagonists arecontemplated as methods and materials of the invention.

EXAMPLE 30 Expression of VEGF-C and FLT4 in the Developing Mouse

Embryos from a 16-day post-coitus pregnant mouse were prepared and fixedin 4% paraformaldehyde (PFA), embedded in paraffin, and sectioned at 6μm. The sections were placed on silanated microscope slides and treatedwith xylene, rehydrated, fixed for 20 minutes in 4% PFA, treated withproteinase K (7 mg/ml; Merck, Darmstadt, Germany) for 5 minutes at roomtemperature, again fixed in 4% PFA and treated with acetic anhydride,dehydrated in solutions with increasing ethanol concentrations, driedand used for in situ hybridization.

In situ hybridization of sections was performed as described (Västrik etal., J. Cell Biol., 128:1197-1208 (1995)). A mouse VEGF-C antisense RNAprobe was generated from linearized pBluescript II SK+ plasmid(Stratagene Inc.), containing a fragment corresponding to nucleotides499-979 of mouse VEGF-C cDNA, where the noncoding region and the BR3Prepeat were removed by Exonuclease III treatment. The fragment had beencloned into the EcoRI and HindIII sites of pBluescript II SK+.Radiolabeled RNA was synthesized using T7 RNA Polymerase and [³⁵S]-UTP(Amersham, Little Chalfont, UK). About two million cpm of the VEGF-Cprobe was applied per slide. After an overnight hybridization, theslides were washed first in 2×SSC and 20-30 mM DDT for 1 hour at 50° C.Treatment continued with a high stringency wash, 4×SSC and 20 mM DTT and50% deionized formamide for 30 minutes at 65° C. followed by RNase Atreatment (20 μg/ml) for 30 minutes at 37° C. The high stringency washwas repeated for 45 minutes. Finally, the slides were dehydrated anddried for 30 minutes at room temperature. The slides were dipped intophotography emulsion and exposed for 4 weeks. Slides were developedusing Kodak D-16 developer, counterstained with hematoxylin and mountedwith Permount (FisherChemical).

For in situ hybridizations of Flt4 sequences, a mouse Flt4 cDNA fragmentcovering bp 1-192 of the published sequence (Finnerty et al., Oncogene,8:2293-2298 (1993)) was used, and the above-described protocol wasfollowed, with the following exceptions. Approximately one million cpmof the Flt4 probe were applied to each slide. The stringent washesfollowing hybridization were performed in 1×SSC and 30 mM DTT for 105minutes.

The figure shows photomicrographs of the hybridized sections in darkfield microscopy (FIGS. 36A-C) and light field microscopy (FIG. 36D).Magnifications used for photography were 4× for FIGS. 36A-B and 10× forFIGS. 36C-D. The transverse sections shown are from the cephalic regionand the area shown for VEGF-C and FLT4 are about 14 sections apart, FLT4being more cranially located in the embryo. In FIG. 36A (Flt4 probe),the developing nasopharyngeal cavity is in the midline in the upper,posterior part; in the anterior part of FIG. 36A is the snout withemerging fibrissal follicles and, in the midline, the forming nasalcavity. On both sides, the retinal pigment gives a false positive signalin dark field microscopy. The most prominently FLT4-hybridizingstructures appear to correspond to the developing lymphatic and venousendothelium. Note that a plexus-like endothelial vascular structuresurrounds the developing nasopharyngeal mucous membrane. In FIG. 36B,the most prominent signal is obtained from the posterior part of thedeveloping nasal conchae, which in higher magnification (FIGS. 36C-D)show the epithelium surrounding loose connective tissue/formingcartilage. This structure gives a strong in situ hybridization signalfor VEGF-C. Also in FIG. 36B, more weakly hybridizing areas can be seenaround the snout, although this signal is much more homogeneous inappearance. Thus, the expression of VEGF-C is strikingly high in thedeveloping nasal conchae.

The conchae are surrounded with a rich vascular plexus, important innasal physiology as a source for the mucus produced by the epithelialcells and for warming inhaled air. It is suggested that VEGF-C isimportant in the formation of the concheal venous plexus at the mucousmembranes, and that it may also regulate the permeability of the vesselsneeded for the secretion of nasal mucus. Possibly, VEGF-C and itsderivatives, and antagonists, could be used in the regulation of theturgor of the conchal tissue and mucous membranes and therefore thediameter of the upper respiratory tract, as well as the quantity andquality of mucus produced. These factors are of great clinicalsignificance in inflammatory (including allergic) and infectiousdiseases of the upper respiratory tract. Accordingly, the inventioncontemplates the use of the materials of the invention, includingVEGF-C, Flt4, and their derivatives, in methods of diagnosing andtreating inflammatory and infectious conditions affecting the upperrespiratory tract, including nasal structures.

DEPOSIT OF BIOLOGICAL MATERIALS

Plasmid FLT4-L has been deposited with the American Type CultureCollection (ATCC), 12301 Parklawn Dr., Rockville Md. 20952 (USA),pursuant to the provisions of the Budapest Treaty, and has been assigneda deposit date of Jul. 24, 1995 and ATCC accession number 97231.

While the present invention has been described in terms of specificembodiments, it is understood that variations and modifications willoccur to those in the art. Accordingly, only such limitations as appearin the appended claims should be placed on the invention.

58 20 base pairs nucleic acid single linear cDNA 1 TGTCCTCGCT GTCCTTGTCT20 70 base pairs nucleic acid single linear cDNA 2 ACATGCATGC CACCATGCAGCGGGGCGCCG CGCTGTGCCT GCGACTGTGG CTCTGCCTGG 60 GACTCCTGGA 70 24 basepairs nucleic acid single linear cDNA 3 ACATGCATGC CCCGCCGGTC ATCC 24 22base pairs nucleic acid single linear cDNA 4 CGGAATTCCC CATGACCCCA AC 2233 base pairs nucleic acid single linear cDNA 5 CCATCGATGG ATCCTACCTGAAGCCGCTTT CTT 33 17 base pairs nucleic acid single linear cDNA 6ATTTAGGTGA CACTATA 17 34 base pairs nucleic acid single linear cDNA 7CCATCGATGG ATCCCGATGC TGCTTAGTAG CTGT 34 40 amino acids amino acidsingle linear peptide 8 Pro Met Thr Pro Thr Thr Tyr Lys Gly Ser Val AspAsn Gln Thr Asp 1 5 10 15 Ser Gly Met Val Leu Ala Ser Glu Glu Phe GluGln Ile Glu Ser Arg 20 25 30 His Arg Gln Glu Ser Gly Phe Arg 35 40 21base pairs nucleic acid single linear cDNA 9 CTGGAGTCGA CTTGGCGGAC T 2160 base pairs nucleic acid single linear cDNA 10 CGCGGATCCC TAGTGATGGTGATGGTGATG TCTACCTTCG ATCATGCTGC CCTTATCCTC 60 34 base pairs nucleicacid single linear cDNA 11 CCCAAGCTTG GATCCAAGTG GCTACTCCAT GACC 34 20base pairs nucleic acid single linear cDNA 12 GTTGCCTGTG ATGTGCACCA 2018 amino acids amino acid single linear peptide 13 Xaa Glu Glu Thr IleLys Phe Ala Ala Ala His Tyr Asn Thr Glu Ile 1 5 10 15 Leu Lys 17 basepairs nucleic acid single linear cDNA 14 GCAGARGARA CNATHAA 17 5 aminoacids amino acid single linear peptide 15 Glu Glu Thr Ile Lys 1 5 18base pairs nucleic acid single linear cDNA 16 GCAYTTNARD ATYTCNGT 18 5amino acids amino acid single linear peptide 17 Thr Glu Ile Leu Lys 1 522 base pairs nucleic acid single linear cDNA 18 ATTCGCTGCA GCACACTACAAC 22 19 base pairs nucleic acid single linear cDNA 19 TCNGTGTTGTAGTGTGCTG 19 7 amino acids amino acid single linear peptide 20 Ala AlaHis Tyr Asn Thr Glu 1 5 20 base pairs nucleic acid single linear cDNA 21TAATACGACT CACTATAGGG 20 24 base pairs nucleic acid single linear cDNA22 GTTGTAGTGT GCTGCAGCGA ATTT 24 8 amino acids amino acid single linearpeptide 23 Lys Phe Ala Ala Ala His Tyr Asn 1 5 21 base pairs nucleicacid single linear cDNA 24 TCACTATAGG GAGACCCAAG C 21 219 base pairsnucleic acid single linear cDNA 25 TCACTATAGG GAGACCCAAG CTTGGTACCGAGCTCGGATC CACTAGTAAC GGCCGCCAGT 60 GTGGTGGAAT TCGACGAACT CATGACTGTACTCTACCCAG AATATTGGAA AATGTACAAG 120 TGTCAGCTAA GGCAAGGAGG CTGGCAACATAACAGAGAAC AGGCCAACCT CAACTCAAGG 180 ACAGAAGAGA CTATAAAATT CGCTGCAGCACACTACAAC 219 18 base pairs nucleic acid single linear cDNA 26ACAGAGAACA GGCCAACC 18 19 base pairs nucleic acid single linear cDNA 27TCTAGCATTT AGGTGACAC 19 25 base pairs nucleic acid single linear cDNA 28AAGAGACTAT AAAATTCGCT GCAGC 25 20 base pairs nucleic acid single linearcDNA 29 CCCTCTAGAT GCATGCTCGA 20 24 base pairs nucleic acid singlelinear cDNA 30 GTTGTAGTGT GCTGCAGCGA ATTT 24 21 base pairs nucleic acidsingle linear cDNA 31 TCACTATAGG GAGACCCAAG C 21 1997 base pairs nucleicacid single linear cDNA CDS 352..1608 32 CCCGCCCCGC CTCTCCAAAAAGCTACACCG ACGCGGACCG CGGCGGCGTC CTCCCTCGCC 60 CTCGCTTCAC CTCGCGGGCTCCGAATGCGG GGAGCTCGGA TGTCCGGTTT CCTGTGAGGC 120 TTTTACCTGA CACCCGCCGCCTTTCCCCGG CACTGGCTGG GAGGGCGCCC TGCAAAGTTG 180 GGAACGCGGA GCCCCGGACCCGCTCCCGCC GCCTCCGGCT CGCCCAGGGG GGGTCGCCGG 240 GAGGAGCCCG GGGGAGAGGGACCAGGAGGG GCCCGCGGCC TCGCAGGGGC GCCCGCGCCC 300 CCACCCCTGC CCCCGCCAGCGGACCGGTCC CCCACCCCCG GTCCTTCCAC C ATG CAC 357 Met His 1 TTG CTG GGC TTCTTC TCT GTG GCG TGT TCT CTG CTC GCC GCT GCG CTG 405 Leu Leu Gly Phe PheSer Val Ala Cys Ser Leu Leu Ala Ala Ala Leu 5 10 15 CTC CCG GGT CCT CGCGAG GCG CCC GCC GCC GCC GCC GCC TTC GAG TCC 453 Leu Pro Gly Pro Arg GluAla Pro Ala Ala Ala Ala Ala Phe Glu Ser 20 25 30 GGA CTC GAC CTC TCG GACGCG GAG CCC GAC GCG GGC GAG GCC ACG GCT 501 Gly Leu Asp Leu Ser Asp AlaGlu Pro Asp Ala Gly Glu Ala Thr Ala 35 40 45 50 TAT GCA AGC AAA GAT CTGGAG GAG CAG TTA CGG TCT GTG TCC AGT GTA 549 Tyr Ala Ser Lys Asp Leu GluGlu Gln Leu Arg Ser Val Ser Ser Val 55 60 65 GAT GAA CTC ATG ACT GTA CTCTAC CCA GAA TAT TGG AAA ATG TAC AAG 597 Asp Glu Leu Met Thr Val Leu TyrPro Glu Tyr Trp Lys Met Tyr Lys 70 75 80 TGT CAG CTA AGG AAA GGA GGC TGGCAA CAT AAC AGA GAA CAG GCC AAC 645 Cys Gln Leu Arg Lys Gly Gly Trp GlnHis Asn Arg Glu Gln Ala Asn 85 90 95 CTC AAC TCA AGG ACA GAA GAG ACT ATAAAA TTT GCT GCA GCA CAT TAT 693 Leu Asn Ser Arg Thr Glu Glu Thr Ile LysPhe Ala Ala Ala His Tyr 100 105 110 AAT ACA GAG ATC TTG AAA AGT ATT GATAAT GAG TGG AGA AAG ACT CAA 741 Asn Thr Glu Ile Leu Lys Ser Ile Asp AsnGlu Trp Arg Lys Thr Gln 115 120 125 130 TGC ATG CCA CGG GAG GTG TGT ATAGAT GTG GGG AAG GAG TTT GGA GTC 789 Cys Met Pro Arg Glu Val Cys Ile AspVal Gly Lys Glu Phe Gly Val 135 140 145 GCG ACA AAC ACC TTC TTT AAA CCTCCA TGT GTG TCC GTC TAC AGA TGT 837 Ala Thr Asn Thr Phe Phe Lys Pro ProCys Val Ser Val Tyr Arg Cys 150 155 160 GGG GGT TGC TGC AAT AGT GAG GGGCTG CAG TGC ATG AAC ACC AGC ACG 885 Gly Gly Cys Cys Asn Ser Glu Gly LeuGln Cys Met Asn Thr Ser Thr 165 170 175 AGC TAC CTC AGC AAG ACG TTA TTTGAA ATT ACA GTG CCT CTC TCT CAA 933 Ser Tyr Leu Ser Lys Thr Leu Phe GluIle Thr Val Pro Leu Ser Gln 180 185 190 GGC CCC AAA CCA GTA ACA ATC AGTTTT GCC AAT CAC ACT TCC TGC CGA 981 Gly Pro Lys Pro Val Thr Ile Ser PheAla Asn His Thr Ser Cys Arg 195 200 205 210 TGC ATG TCT AAA CTG GAT GTTTAC AGA CAA GTT CAT TCC ATT ATT AGA 1029 Cys Met Ser Lys Leu Asp Val TyrArg Gln Val His Ser Ile Ile Arg 215 220 225 CGT TCC CTG CCA GCA ACA CTACCA CAG TGT CAG GCA GCG AAC AAG ACC 1077 Arg Ser Leu Pro Ala Thr Leu ProGln Cys Gln Ala Ala Asn Lys Thr 230 235 240 TGC CCC ACC AAT TAC ATG TGGAAT AAT CAC ATC TGC AGA TGC CTG GCT 1125 Cys Pro Thr Asn Tyr Met Trp AsnAsn His Ile Cys Arg Cys Leu Ala 245 250 255 CAG GAA GAT TTT ATG TTT TCCTCG GAT GCT GGA GAT GAC TCA ACA GAT 1173 Gln Glu Asp Phe Met Phe Ser SerAsp Ala Gly Asp Asp Ser Thr Asp 260 265 270 GGA TTC CAT GAC ATC TGT GGACCA AAC AAG GAG CTG GAT GAA GAG ACC 1221 Gly Phe His Asp Ile Cys Gly ProAsn Lys Glu Leu Asp Glu Glu Thr 275 280 285 290 TGT CAG TGT GTC TGC AGAGCG GGG CTT CGG CCT GCC AGC TGT GGA CCC 1269 Cys Gln Cys Val Cys Arg AlaGly Leu Arg Pro Ala Ser Cys Gly Pro 295 300 305 CAC AAA GAA CTA GAC AGAAAC TCA TGC CAG TGT GTC TGT AAA AAC AAA 1317 His Lys Glu Leu Asp Arg AsnSer Cys Gln Cys Val Cys Lys Asn Lys 310 315 320 CTC TTC CCC AGC CAA TGTGGG GCC AAC CGA GAA TTT GAT GAA AAC ACA 1365 Leu Phe Pro Ser Gln Cys GlyAla Asn Arg Glu Phe Asp Glu Asn Thr 325 330 335 TGC CAG TGT GTA TGT AAAAGA ACC TGC CCC AGA AAT CAA CCC CTA AAT 1413 Cys Gln Cys Val Cys Lys ArgThr Cys Pro Arg Asn Gln Pro Leu Asn 340 345 350 CCT GGA AAA TGT GCC TGTGAA TGT ACA GAA AGT CCA CAG AAA TGC TTG 1461 Pro Gly Lys Cys Ala Cys GluCys Thr Glu Ser Pro Gln Lys Cys Leu 355 360 365 370 TTA AAA GGA AAG AAGTTC CAC CAC CAA ACA TGC AGC TGT TAC AGA CGG 1509 Leu Lys Gly Lys Lys PheHis His Gln Thr Cys Ser Cys Tyr Arg Arg 375 380 385 CCA TGT ACG AAC CGCCAG AAG GCT TGT GAG CCA GGA TTT TCA TAT AGT 1557 Pro Cys Thr Asn Arg GlnLys Ala Cys Glu Pro Gly Phe Ser Tyr Ser 390 395 400 GAA GAA GTG TGT CGTTGT GTC CCT TCA TAT TGG AAA AGA CCA CAA ATG 1605 Glu Glu Val Cys Arg CysVal Pro Ser Tyr Trp Lys Arg Pro Gln Met 405 410 415 AGC TAAGATTGTACTGTTTTCCA GTTCATCGAT TTTCTATTAT GGAAAACTGT 1658 Ser GTTGCCACAGTAGAACTGTC TGTGAACAGA GAGACCCTTG TGGGTCCATG CTAACAAAGA 1718 CAAAAGTCTGTCTTTCCTGA ACCATGTGGA TAACTTTACA GAAATGGACT GGAGCTCATC 1778 TGCAAAAGGCCTCTTGTAAA GACTGGTTTT CTGCCAATGA CCAAACAGCC AAGATTTTCC 1838 TCTTGTGATTTCTTTAAAAG AATGACTATA TAATTTATTT CCACTAAAAA TATTGTTTCT 1898 GCATTCATTTTTATAGCAAC AACAATTGGT AAAACTCACT GTGATCAATA TTTTTATATC 1958 ATGCAAAATATGTTTAAAAT AAAATGAAAA TTGTATTAT 1997 419 amino acids amino acid linearprotein 33 Met His Leu Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu AlaAla 1 5 10 15 Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala AlaAla Phe 20 25 30 Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala GlyGlu Ala 35 40 45 Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu Gln Leu Arg SerVal Ser 50 55 60 Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr TrpLys Met 65 70 75 80 Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His AsnArg Glu Gln 85 90 95 Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys PheAla Ala Ala 100 105 110 His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp AsnGlu Trp Arg Lys 115 120 125 Thr Gln Cys Met Pro Arg Glu Val Cys Ile AspVal Gly Lys Glu Phe 130 135 140 Gly Val Ala Thr Asn Thr Phe Phe Lys ProPro Cys Val Ser Val Tyr 145 150 155 160 Arg Cys Gly Gly Cys Cys Asn SerGlu Gly Leu Gln Cys Met Asn Thr 165 170 175 Ser Thr Ser Tyr Leu Ser LysThr Leu Phe Glu Ile Thr Val Pro Leu 180 185 190 Ser Gln Gly Pro Lys ProVal Thr Ile Ser Phe Ala Asn His Thr Ser 195 200 205 Cys Arg Cys Met SerLys Leu Asp Val Tyr Arg Gln Val His Ser Ile 210 215 220 Ile Arg Arg SerLeu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn 225 230 235 240 Lys ThrCys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys 245 250 255 LeuAla Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser 260 265 270Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu 275 280285 Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys 290295 300 Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys305 310 315 320 Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu PheAsp Glu 325 330 335 Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys Pro ArgAsn Gln Pro 340 345 350 Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr GluSer Pro Gln Lys 355 360 365 Cys Leu Leu Lys Gly Lys Lys Phe His His GlnThr Cys Ser Cys Tyr 370 375 380 Arg Arg Pro Cys Thr Asn Arg Gln Lys AlaCys Glu Pro Gly Phe Ser 385 390 395 400 Tyr Ser Glu Glu Val Cys Arg CysVal Pro Ser Tyr Trp Lys Arg Pro 405 410 415 Gln Met Ser 22 base pairsnucleic acid single linear cDNA 34 TGAGTGATTTGTAGCTGCTGTG 22 22 basepairs nucleic acid single linear cDNA 35 TATTGCAGCAACCCCCACATCT 22 4416base pairs nucleic acid single linear cDNA 36 CCACGCGCAG CGGCCGGAGATGCAGCGGGG CGCCGCGCTG TGCCTGCGAC TGTGGCTCTG 60 CCTGGGACTC CTGGACGGCCTGGTGAGTGG CTACTCCATG ACCCCCCCGA CCTTGAACAT 120 CACGGAGGAG TCACACGTCATCGACACCGG TGACAGCCTG TCCATCTCCT GCAGGGGACA 180 GCACCCCCTC GAGTGGGCTTGGCCAGGAGC TCAGGAGGCG CCAGCCACCG GAGACAAGGA 240 CAGCGAGGAC ACGGGGGTGGTGCGAGACTG CGAGGGCACA GACGCCAGGC CCTACTGCAA 300 GGTGTTGCTG CTGCACGAGGTACATGCCAA CGACACAGGC AGCTACGTCT GCTACTACAA 360 GTACATCAAG GCACGCATCGAGGGCACCAC GGCCGCCAGC TCCTACGTGT TCGTGAGAGA 420 CTTTGAGCAG CCATTCATCAACAAGCCTGA CACGCTCTTG GTCAACAGGA AGGACGCCAT 480 GTGGGTGCCC TGTCTGGTGTCCATCCCCGG CCTCAATGTC ACGCTGCGCT CGCAAAGCTC 540 GGTGCTGTGG CCAGACGGGCAGGAGGTGGT GTGGGATGAC CGGCGGGGCA TGCTCGTGTC 600 CACGCCACTG CTGCACGATGCCCTGTACCT GCAGTGCGAG ACCACCTGGG GAGACCAGGA 660 CTTCCTTTCC AACCCCTTCCTGGTGCACAT CACAGGCAAC GAGCTCTATG ACATCCAGCT 720 GTTGCCCAGG AAGTCGCTGGAGCTGCTGGT AGGGGAGAAG CTGGTCCTGA ACTGCACCGT 780 GTGGGCTGAG TTTAACTCAGGTGTCACCTT TGACTGGGAC TACCCAGGGA AGCAGGCAGA 840 GCGGGGTAAG TGGGTGCCCGAGCGACGCTC CCAGCAGACC CACACAGAAC TCTCCAGCAT 900 CCTGACCATC CACAACGTCAGCCAGCACGA CCTGGGCTCG TATGTGTGCA AGGCCAACAA 960 CGGCATCCAG CGATTTCGGGAGAGCACCGA GGTCATTGTG CATGAAAATC CCTTCATCAG 1020 CGTCGAGTGG CTCAAAGGACCCATCCTGGA GGCCACGGCA GGAGACGAGC TGGTGAAGCT 1080 GCCCGTGAAG CTGGCAGCGTACCCCCCGCC CGAGTTCCAG TGGTACAAGG ATGGAAAGGC 1140 ACTGTCCGGG CGCCACAGTCCACATGCCCT GGTGCTCAAG GAGGTGACAG AGGCCAGCAC 1200 AGGCACCTAC ACCCTCGCCCTGTGGAACTC CGCTGCTGGC CTGAGGCGCA ACATCAGCCT 1260 GGAGCTGGTG GTGAATGTGCCCCCCCAGAT ACATGAGAAG GAGGCCTCCT CCCCCAGCAT 1320 CTACTCGCGT CACAGCCGCCAGGCCCTCAC CTGCACGGCC TACGGGGTGC CCCTGCCTCT 1380 CAGCATCCAG TGGCACTGGCGGCCCTGGAC ACCCTGCAAG ATGTTTGCCC AGCGTAGTCT 1440 CCGGCGGCGG CAGCAGCAAGACCTCATGCC ACAGTGCCGT GACTGGAGGG CGGTGACCAC 1500 GCAGGATGCC GTGAACCCCATCGAGAGCCT GGACACCTGG ACCGAGTTTG TGGAGGGAAA 1560 GAATAAGACT GTGAGCAAGCTGGTGATCCA GAATGCCAAC GTGTCTGCCA TGTACAAGTG 1620 TGTGGTCTCC AACAAGGTGGGCCAGGATGA GCGGCTCATC TACTTCTATG TGACCACCAT 1680 CCCCGACGGC TTCACCATCGAATCCAAGCC ATCCGAGGAG CTACTAGAGG GCCAGCCGGT 1740 GCTCCTGAGC TGCCAAGCCGACAGCTACAA GTACGAGCAT CTGCGCTGGT ACCGCCTCAA 1800 CCTGTCCACG CTGCACGATGCGCACGGGAA CCCGCTTCTG CTCGACTGCA AGAACGTGCA 1860 TCTGTTCGCC ACCCCTCTGGCCGCCAGCCT GGAGGAGGTG GCACCTGGGG CGCGCCACGC 1920 CACGCTCAGC CTGAGTATCCCCCGCGTCGC GCCCGAGCAC GAGGGCCACT ATGTGTGCGA 1980 AGTGCAAGAC CGGCGCAGCCATGACAAGCA CTGCCACAAG AAGTACCTGT CGGTGCAGGC 2040 CCTGGAAGCC CCTCGGCTCACGCAGAACTT GACCGACCTC CTGGTGAACG TGAGCGACTC 2100 GCTGGAGATG CAGTGCTTGGTGGCCGGAGC GCACGCGCCC AGCATCGTGT GGTACAAAGA 2160 CGAGAGGCTG CTGGAGGAAAAGTCTGGAGT CGACTTGGCG GACTCCAACC AGAAGCTGAG 2220 CATCCAGCGC GTGCGCGAGGAGGATGCGGG ACGCTATCTG TGCAGCGTGT GCAACGCCAA 2280 GGGCTGCGTC AACTCCTCCGCCAGCGTGGC CGTGGAAGGC TCCGAGGATA AGGGCAGCAT 2340 GGAGATCGTG ATCCTTGTCGGTACCGGCGT CATCGCTGTC TTCTTCTGGG TCCTCCTCCT 2400 CCTCATCTTC TGTAACATGAGGAGGCCGGC CCACGCAGAC ATCAAGACGG GCTACCTGTC 2460 CATCATCATG GACCCCGGGGAGGTGCCTCT GGAGGAGCAA TGCGAATACC TGTCCTACGA 2520 TGCCAGCCAG TGGGAATTCCCCCGAGAGCG GCTGCACCTG GGGAGAGTGC TCGGCTACGG 2580 CGCCTTCGGG AAGGTGGTGGAAGCCTCCGC TTTCGGCATC CACAAGGGCA GCAGCTGTGA 2640 CACCGTGGCC GTGAAAATGCTGAAAGAGGG CGCCACGGCC AGCGAGCACC GCGCGCTGAT 2700 GTCGGAGCTC AAGATCCTCATTCACATCGG CAACCACCTC AACGTGGTCA ACCTCCTCGG 2760 GGCGTGCACC AAGCCGCAGGGCCCCCTCAT GGTGATCGTG GAGTTCTGCA AGTACGGCAA 2820 CCTCTCCAAC TTCCTGCGCGCCAAGCGGGA CGCCTTCAGC CCCTGCGCGG AGAAGTCTCC 2880 CGAGCAGCGC GGACGCTTCCGCGCCATGGT GGAGCTCGCC AGGCTGGATC GGAGGCGGCC 2940 GGGGAGCAGC GACAGGGTCCTCTTCGCGCG GTTCTCGAAG ACCGAGGGCG GAGCGAGGCG 3000 GGCTTCTCCA GACCAAGAAGCTGAGGACCT GTGGCTGAGC CCGCTGACCA TGGAAGATCT 3060 TGTCTGCTAC AGCTTCCAGGTGGCCAGAGG GATGGAGTTC CTGGCTTCCC GAAAGTGCAT 3120 CCACAGAGAC CTGGCTGCTCGGAACATTCT GCTGTCGGAA AGCGACGTGG TGAAGATCTG 3180 TGACTTTGGC CTTGCCCGGGACATCTACAA AGACCCTGAC TACGTCCGCA AGGGCAGTGC 3240 CCGGCTGCCC CTGAAGTGGATGGCCCCTGA AAGCATCTTC GACAAGGTGT ACACCACGCA 3300 GAGTGACGTG TGGTCCTTTGGGGTGCTTCT CTGGGAGATC TTCTCTCTGG GGGCCTCCCC 3360 GTACCCTGGG GTGCAGATCAATGAGGAGTT CTGCCAGCGG CTGAGAGACG GCACAAGGAT 3420 GAGGGCCCCG GAGCTGGCCACTCCCGCCAT ACGCCGCATC ATGCTGAACT GCTGGTCCGG 3480 AGACCCCAAG GCGAGACCTGCATTCTCGGA GCTGGTGGAG ATCCTGGGGG ACCTGCTCCA 3540 GGGCAGGGGC CTGCAAGAGGAAGAGGAGGT CTGCATGGCC CCGCGCAGCT CTCAGAGCTC 3600 AGAAGAGGGC AGCTTCTCGCAGGTGTCCAC CATGGCCCTA CACATCGCCC AGGCTGACGC 3660 TGAGGACAGC CCGCCAAGCCTGCAGCGCCA CAGCCTGGCC GCCAGGTATT ACAACTGGGT 3720 GTCCTTTCCC GGGTGCCTGGCCAGAGGGGC TGAGACCCGT GGTTCCTCCA GGATGAAGAC 3780 ATTTGAGGAA TTCCCCATGACCCCAACGAC CTACAAAGGC TCTGTGGACA ACCAGACAGA 3840 CAGTGGGATG GTGCTGGCCTCGGAGGAGTT TGAGCAGATA GAGAGCAGGC ATAGACAAGA 3900 AAGCGGCTTC AGGTAGCTGAAGCAGAGAGA GAGAAGGCAG CATACGTCAG CATTTTCTTC 3960 TCTGCACTTA TAAGAAAGATCAAAGACTTT AAGACTTTCG CTATTTCTTC TACTGCTATC 4020 TACTACAAAC TTCAAAGAGGAACCAGGAGG ACAAGAGGAG CATGAAAGTG GACAAGGAGT 4080 GTGACCACTG AAGCACCACAGGGAAGGGGT TAGGCCTCCG GATGACTGCG GGCAGGCCTG 4140 GATAATATCC AGCCTCCCACAAGAAGCTGG TGGAGCAGAG TGTTCCCTGA CTCCTCCAAG 4200 GAAAGGGAGA CGCCCTTTCATGGTCTGCTG AGTAACAGGT GCNTTCCCAG ACACTGGCGT 4260 TACTGCTTGA CCAAAGAGCCCTCAAGCGGC CCTTATGCCA GCGTGACAGA GGGCTCACCT 4320 CTTGCCTTCT AGGTCACTTCTCACACAATG TCCCTTCAGC ACCTGACCCT GTGCCCGCCA 4380 GTTATTCCTT GGTAATATGAGTAATACATC AAAGAG 4416 4273 base pairs nucleic acid single linear cDNA37 AAGCTTATCG ATTTCGAACC CGGGGGTACC GAATTCCTCG AGTCTAGAGG AGCATGCCTG 60CAGGTCGACC GGGCTCGATC CCCTCGCGAG TTGGTTCAGC TGCTGCCTGA GGCTGGACGA 120CCTCGCGGAG TTCTACCGGC AGTGCAAATC CGTCGGCATC CAGGAAACCA GCAGCGGCTA 180TCCGCGCATC CATGCCCCCG AACTGCAGGA GTGGGGAGGC ACGATGGCCG CTTTGGTCCC 240GGATCTTTGT GAAGGAACCT TACTTCTGTG GTGTGACATA ATTGGACAAA CTACCTACAG 300AGATTTAAAG CTCTAAGGTA AATATAAAAT TTTTAAGTGT ATAATGTGTT AAACTACTGA 360TTCTAATTGT TTGTGTATTT TAGATTCCAA CCTATGGAAC TGATGAATGG GAGCAGTGGT 420GGAATGCCTT TAATGAGGAA AACCTGTTTT GCTCAGAAGA AATGCCATCT AGTGATGATG 480AGGCTACTGC TGACTCTCAA CATTCTACTC CTCCAAAAAA GAAGAGAAAG GTAGAAGACC 540CCAAGGACTT TCCTTCAGAA TTGCTAAGTT TTTTGAGTCA TGCTGTGTTT AGTAATAGAA 600CTCTTGCTTG CTTTGCTATT TACACCACAA AGGAAAAAGC TGCACTGCTA TACAAGAAAA 660TTATGGAAAA ATATTCTGTA ACCTTTATAA GTAGGCATAA CAGTTATAAT CATAACATAC 720TGTTTTTTCT TACTCCACAC AGGCATAGAG TGTCTGCTAT TAATAACTAT GCTCAAAAAT 780TGTGTACCTT TAGCTTTTTA ATTTGTAAAG GGGTTAATAA GGAATATTTG ATGTATAGTG 840CCTTGACTAG AGATCATAAT CAGCCATACC ACATTTGTAG AGGTTTTACT TGCTTTAAAA 900AACCTCCCAC ACCTCCCCCT GAACCTGAAA CATAAAATGA ATGCAATTGT TGTTGTTAAC 960TTGTTTATTG CAGCTTATAA TGGTTACAAA TAAAGCAATA GCATCACAAA TTTCACAAAT 1020AAAGCATTTT TTTCACTGCA TTCTAGTTGT GGTTTGTCCA AACTCATCAA TGTATCTTAT 1080CATGTCTGGA TCTGCCGGTC TCCCTATAGT GAGTCGTATT AATTTCGATA AGCCAGGTTA 1140ACCTGCATTA ATGAATCGGC CAACGCGCGG GGAGAGGCGG TTTGCGTATT GGGCGCTCTT 1200CCGCTTCCTC GCTCACTGAC TCGCTGCGCT CGGTCGTTCG GCTGCGGCGA GCGGTATCAG 1260CTCACTCAAA GGCGGTAATA CGGTTATCCA CAGAATCAGG GGATAACGCA GGAAAGAACA 1320TGTGAGCAAA AGGCCAGCAA AAGGCCAGGA ACCGTAAAAA GGACGCGTTG CTGGCGTTTT 1380TCCATAGGCT CCGCCCCCCT GACGAGCATC ACAAAAATCG ACGCTCAAGT CAGAGGTGGC 1440GAAACCCGAC AGGACTATAA AGATACCAGG CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT 1500CTCCTGTTCC GACCCTGCCG CTTACCGGAT ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG 1560TGGCGCTTTC TCAATGCTCA CGCTGTAGGT ATCTCAGTTC GGTGTAGGTC GTTCGCTCCA 1620AGCTGGGCTG TGTGCACGAA CCCCCCGTTC AGCCCGACCG CTGCGCCTTA TCCGGTAACT 1680ATCGTCTTGA GTCCAACCCG GTAAGACACG ACTTATCGCC ACTGGCAGCA GCCACTGGTA 1740ACAGGATTAG CAGAGCGAGG TATGTAGGCG GTGCTACAGA GTTCTTGAAG TGGTGGCCTA 1800ACTACGGCTA CACTAGAAGG ACAGTATTTG GTATCTGCGC TCTGCTGAAG CCAGTTACCT 1860TCGGAAAAAG AGTTGGTAGC TCTTGATCCG GCAAACAAAC CACCGCTGGT AGCGGTGGTT 1920TTTTTGTTTG CAAGCAGCAG ATTACGCGCA GAAAAAAAGG ATCTCAAGAA GATCCTTTGA 1980TCTTTTCTAC GGGGTCTGAC GCTCAGTGGA ACGAAAACTC ACGTTAAGGG ATTTTGGTCA 2040TGAGATTATC AAAAAGGATC TTCACCTAGA TCCTTTTAAA TTAAAAATGA AGTTTTAAAT 2100CAATCTAAAG TATATATGAG TAAACTTGGT CTGACAGTTA CCAATGCTTA ATCAGTGAGG 2160CACCTATCTC AGCGATCTGT CTATTTCGTT CATCCATAGT TGCCTGACTC CCCGTCGTGT 2220AGATAACTAC GATACGGGAG GGCTTACCAT CTGGCCCCAG TGCTGCAATG ATACCGCGAG 2280ACCCACGCTC ACCGGCTCCA GATTTATCAG CAATAAACCA GCCAGCCGGA AGGGCCGAGC 2340GCAGAAGTGG TCCTGCAACT TTATCCGCCT CCATCCAGTC TATTAATTGT TGCCGGGAAG 2400CTAGAGTAAG TAGTTCGCCA GTTAATAGTT TGCGCAACGT TGTTGCCATT GCTACAGGCA 2460TCGTGGTGTC ACGCTCGTCG TTTGGTATGG CTTCATTCAG CTCCGGTTCC CAACGATCAA 2520GGCGAGTTAC ATGATCCCCC ATGTTGTGCA AAAAAGCGGT TAGCTCCTTC GGTCCTCCGA 2580TCGTTGTCAG AAGTAAGTTG GCCGCAGTGT TATCACTCAT GGTTATGGCA GCACTGCATA 2640ATTCTCTTAC TGTCATGCCA TCCGTAAGAT GCTTTTCTGT GACTGGTGAG TACTCAACCA 2700AGTCATTCTG AGAATAGTGT ATGCGGCGAC CGAGTTGCTC TTGCCCGGCG TCAATACGGG 2760ATAATACCGC GCCACATAGC AGAACTTTAA AAGTGCTCAT CATTGGAAAA CGTTCTTCGG 2820GGCGAAAACT CTCAAGGATC TTACCGCTGT TGAGATCCAG TTCGATGTAA CCCACTCGTG 2880CACCCAACTG ATCTTCAGCA TCTTTTACTT TCACCAGCGT TTCTGGGTGA GCAAAAACAG 2940GAAGGCAAAA TGCCGCAAAA AAGGGAATAA GGGCGACACG GAAATGTTGA ATACTCATAC 3000TCTTCCTTTT TCAATATTAT TGAAGCATTT ATCAGGGTTA TTGTCTCATG AGCGGATACA 3060TATTTGAATG TATTTAGAAA AATAAACAAA TAGGGGTTCC GCGCACATTT CCCCGAAAAG 3120TGCCACCTGA CGTCTAAGAA ACCATTATTA TCATGACATT AACCTATAAA AATAGGCGTA 3180TCACGAGGCC CTTTCGTCTC GCGCGTTTCG GTGATGACGG TGAAAACCTC TGACACATGC 3240AGCTCCCGGA GACGGTCACA GCTTGTCTGT AAGCGGATGC CGGGAGCAGA CAAGCCCGTC 3300AGGGCGCGTC AGCGGGTGTT GGCGGGTGTC GGGGCTGGCT TAACTATGCG GCATCAGAGC 3360AGATTGTACT GAGAGTGCAC CATATGGACA TATTGTCGTT AGAACGCGGC TACAATTAAT 3420ACATAACCTT ATGTATCATA CACATACGAT TTAGGTGACA CTATAGAACT CGAGCAGAGC 3480TTCCAAATTG AGAGAGAGGC TTAATCAGAG ACAGAAACTG TTTGAGTCAA CTCAAGGATG 3540GTTTGAGGGA CTGTTTAACA GATCCCCTTG GTTTACCACC TTGATATCTA CCATTATGGG 3600ACCCCTCATT GTACTCCTAA TGATTTTGCT CTTCGGACCC TGCATTCTTA ATCGATTAGT 3660CCAATTTGTT AAAGACAGGA TATCAGTGGT CCAGGCTCTA GTTTTGACTC AACAATATCA 3720CCAGCTGAAG CCTATAGAGT ACGAGCCATA GATAAAATAA AAGATTTTAT TTAGTCTCCA 3780GAAAAAGGGG GGAATGAAAG ACCCCACCTG TAGGTTTGGC AAGCTAGCTT AAGTAACGCC 3840ATTTTGCAAG GCATGGAAAA ATACATAACT GAGAATAGAG AAGTTCAGAT CAAGGTCAGG 3900AACAGATGGA ACAGCTGAAT ATGGGCCAAA CAGGATATCT GTGGTAAGCA GTTCCTGCCC 3960CGGCTCAGGG CCAAGAACAG ATGGAACAGC TGAATATGGG CCAAACAGGA TATCTGTGGT 4020AAGCAGTTCC TGCCCCGGCT CAGGGCCAAG AACAGATGGT CCCCAGATGC GGTCCAGCCC 4080TCAGCAGTTT CTAGAGAACC ATCAGATGTT TCCAGGGTGC CCCAAGGACC TGAAATGACC 4140CTGTGCCTTA TTTGAACTAA CCAATCAGTT CGCTTCTCGC TTCTGTTCGC GCGCTTCTGC 4200TCCCCGAGCT CAATAAAAGA GCCCACAACC CCTCACTCGG GGCGCCAGTC CTCCGATTGA 4260CTGAGTCGCC CGG 4273 216 base pairs nucleic acid single linear cDNA 38CAAGAAAGCG GCTTCAGCTG TAAAGGACCT GGCCAGAATG TGGCTGTGAC CAGGGCACAC 60CCTGACTCCC AAGGGAGGCG GCGGCGGCCT GAGCGGGGGG CCCGAGGAGG CCAGGTGTTT 120TACAACAGCG AGTATGGGGA GCTGTCGGAG CCAAGCGAGG AGGACCACTG CTCCCCGTCT 180GCCCGCGTGA CTTTCTTCAC AGACAACAGC TACTAA 216 17 amino acids amino acidsingle linear peptide 39 Glu Glu Thr Ile Lys Phe Ala Ala Ala His Tyr AsnThr Glu Ile Leu 1 5 10 15 Lys 1836 base pairs nucleic acid single linearcDNA CDS 168..1412 40 GCGGCCGCGT CGACGCAAAA GTTGCGAGCC GCCGAGTCCCGGGAGACGCT CGCCCAGGGG 60 GGTCCCCGGG AGGAAACCAC GGGACAGGGA CCAGGAGAGGACCTCAGCCT CACGCCCCAG 120 CCTGCGCCAG CCAACGGACC GGCCTCCCTG CTCCCGGTCCATCCACC ATG CAC TTG 176 Met His Leu 1 CTG TGC TTC TTG TCT CTG GCG TGTTCC CTG CTC GCC GCT GCG CTG ATC 224 Leu Cys Phe Leu Ser Leu Ala Cys SerLeu Leu Ala Ala Ala Leu Ile 5 10 15 CCC AGT CCG CGC GAG GCG CCC GCC ACCGTC GCC GCC TTC GAG TCG GGA 272 Pro Ser Pro Arg Glu Ala Pro Ala Thr ValAla Ala Phe Glu Ser Gly 20 25 30 35 CTG GGC TTC TCG GAA GCG GAG CCC GACGGG GGC GAG GTC AAG GCT TTT 320 Leu Gly Phe Ser Glu Ala Glu Pro Asp GlyGly Glu Val Lys Ala Phe 40 45 50 GAA GGC AAA GAC CTG GAG GAG CAG TTG CGGTCT GTG TCC AGC GTA GAT 368 Glu Gly Lys Asp Leu Glu Glu Gln Leu Arg SerVal Ser Ser Val Asp 55 60 65 GAG CTG ATG TCT GTC CTG TAC CCA GAC TAC TGGAAA ATG TAC AAG TGC 416 Glu Leu Met Ser Val Leu Tyr Pro Asp Tyr Trp LysMet Tyr Lys Cys 70 75 80 CAG CTG CGG AAA GGC GGC TGG CAG CAG CCC ACC CTCAAT ACC AGG ACA 464 Gln Leu Arg Lys Gly Gly Trp Gln Gln Pro Thr Leu AsnThr Arg Thr 85 90 95 GGG GAC AGT GTA AAA TTT GCT GCT GCA CAT TAT AAC ACAGAG ATC CTG 512 Gly Asp Ser Val Lys Phe Ala Ala Ala His Tyr Asn Thr GluIle Leu 100 105 110 115 AAA AGT ATT GAT AAT GAG TGG AGA AAG ACT CAA TGCATG CCA CGT GAG 560 Lys Ser Ile Asp Asn Glu Trp Arg Lys Thr Gln Cys MetPro Arg Glu 120 125 130 GTG TGT ATA GAT GTG GGG AAG GAG TTT GGA GCA GCCACA AAC ACC TTC 608 Val Cys Ile Asp Val Gly Lys Glu Phe Gly Ala Ala ThrAsn Thr Phe 135 140 145 TTT AAA CCT CCA TGT GTG TCC GTC TAC AGA TGT GGGGGT TGC TGC AAC 656 Phe Lys Pro Pro Cys Val Ser Val Tyr Arg Cys Gly GlyCys Cys Asn 150 155 160 AGG GAG GGG CTG CAG TGC ATG AAC ACC AGC ACA GGTTAC CTC AGC AAG 704 Arg Glu Gly Leu Gln Cys Met Asn Thr Ser Thr Gly TyrLeu Ser Lys 165 170 175 ACG TTG TTT GAA ATT ACA GTG CCT CTC TCA CAA GGCCCC AAA CCA GTC 752 Thr Leu Phe Glu Ile Thr Val Pro Leu Ser Gln Gly ProLys Pro Val 180 185 190 195 ACA ATC AGT TTT GCC AAT CAC ACT TCC TGC CGGTGC ATG TCT AAA CTG 800 Thr Ile Ser Phe Ala Asn His Thr Ser Cys Arg CysMet Ser Lys Leu 200 205 210 GAT GTT TAC AGA CAA GTT CAT TCA ATT ATT AGACGT TCT CTG CCA GCA 848 Asp Val Tyr Arg Gln Val His Ser Ile Ile Arg ArgSer Leu Pro Ala 215 220 225 ACA TTA CCA CAG TGT CAG GCA GCT AAC AAG ACATGT CCA ACA AAC TAT 896 Thr Leu Pro Gln Cys Gln Ala Ala Asn Lys Thr CysPro Thr Asn Tyr 230 235 240 GTG TGG AAT AAC TAC ATG TGC CGA TGC CTG GCTCAG CAG GAT TTT ATC 944 Val Trp Asn Asn Tyr Met Cys Arg Cys Leu Ala GlnGln Asp Phe Ile 245 250 255 TTT TAT TCA AAT GTT GAA GAT GAC TCA ACC AATGGA TTC CAT GAT GTC 992 Phe Tyr Ser Asn Val Glu Asp Asp Ser Thr Asn GlyPhe His Asp Val 260 265 270 275 TGT GGA CCC AAC AAG GAG CTG GAT GAA GACACC TGT CAG TGT GTC TGC 1040 Cys Gly Pro Asn Lys Glu Leu Asp Glu Asp ThrCys Gln Cys Val Cys 280 285 290 AAG GGG GGG CTT CGG CCA TCT AGT TGT GGACCC CAC AAA GAA CTA GAT 1088 Lys Gly Gly Leu Arg Pro Ser Ser Cys Gly ProHis Lys Glu Leu Asp 295 300 305 AGA GAC TCA TGT CAG TGT GTC TGT AAA AACAAA CTT TTC CCT AAT TCA 1136 Arg Asp Ser Cys Gln Cys Val Cys Lys Asn LysLeu Phe Pro Asn Ser 310 315 320 TGT GGA GCC AAC AGG GAA TTT GAT GAG AATACA TGT CAG TGT GTA TGT 1184 Cys Gly Ala Asn Arg Glu Phe Asp Glu Asn ThrCys Gln Cys Val Cys 325 330 335 AAA AGA ACG TGT CCA AGA AAT CAG CCC CTGAAT CCT GGG AAA TGT GCC 1232 Lys Arg Thr Cys Pro Arg Asn Gln Pro Leu AsnPro Gly Lys Cys Ala 340 345 350 355 TGT GAA TGT ACA GAA AAC ACA CAG AAGTGC TTC CTT AAA GGG AAG AAG 1280 Cys Glu Cys Thr Glu Asn Thr Gln Lys CysPhe Leu Lys Gly Lys Lys 360 365 370 TTC CAC CAT CAA ACA TGC AGT TGT TACAGA AGA CCG TGT GCG AAT CGA 1328 Phe His His Gln Thr Cys Ser Cys Tyr ArgArg Pro Cys Ala Asn Arg 375 380 385 CTG AAG CAT TGT GAT CCA GGA CTG TCCTTT AGT GAA GAA GTA TGC CGC 1376 Leu Lys His Cys Asp Pro Gly Leu Ser PheSer Glu Glu Val Cys Arg 390 395 400 TGT GTC CCA TCG TAT TGG AAA AGG CCACAT CTG AAC TAAGATCATA 1422 Cys Val Pro Ser Tyr Trp Lys Arg Pro His LeuAsn 405 410 415 CCAGTTTTCA GTCAGTCACA GTCATTTACT CTCTTGAAGA CTGTTGGAACAGCACTTAGC 1482 ACTGTCTATG CACAGAAAGA CTCTGTGGGA CCACATGGTA ACAGAGGCCCAAGTCTGTGT 1542 TTATTGAACC ATGTGGATTA CTGCGGGAGA GGACTGGCAC TCATGTGCAAAAAAAACCTC 1602 TTCAAAGACT GGTTTTCTGC CAGGGACCAG ACAGCTGAGG TTTTTCTCTTGTGATTTAAA 1662 AAAAGAATGA CTATATAATT TATTTCCACT AAAAATATTG TTCCTGCATTCATTTTTATA 1722 GCAATAACAA TTGGTAAAGC TCACTGTGAT CAGTATTTTT ATAACATGCAAAACTATGTT 1782 TAAAATAAAA TGAAAATTGT ATTATAAAAA AAAAAAAAAA AAAAAAAAAAGCTT 1836 415 amino acids amino acid linear protein 41 Met His Leu LeuCys Phe Leu Ser Leu Ala Cys Ser Leu Leu Ala Ala 1 5 10 15 Ala Leu IlePro Ser Pro Arg Glu Ala Pro Ala Thr Val Ala Ala Phe 20 25 30 Glu Ser GlyLeu Gly Phe Ser Glu Ala Glu Pro Asp Gly Gly Glu Val 35 40 45 Lys Ala PheGlu Gly Lys Asp Leu Glu Glu Gln Leu Arg Ser Val Ser 50 55 60 Ser Val AspGlu Leu Met Ser Val Leu Tyr Pro Asp Tyr Trp Lys Met 65 70 75 80 Tyr LysCys Gln Leu Arg Lys Gly Gly Trp Gln Gln Pro Thr Leu Asn 85 90 95 Thr ArgThr Gly Asp Ser Val Lys Phe Ala Ala Ala His Tyr Asn Thr 100 105 110 GluIle Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys Thr Gln Cys Met 115 120 125Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe Gly Ala Ala Thr 130 135140 Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr Arg Cys Gly Gly 145150 155 160 Cys Cys Asn Arg Glu Gly Leu Gln Cys Met Asn Thr Ser Thr GlyTyr 165 170 175 Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu Ser GlnGly Pro 180 185 190 Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser CysArg Cys Met 195 200 205 Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser IleIle Arg Arg Ser 210 215 220 Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala AlaAsn Lys Thr Cys Pro 225 230 235 240 Thr Asn Tyr Val Trp Asn Asn Tyr MetCys Arg Cys Leu Ala Gln Gln 245 250 255 Asp Phe Ile Phe Tyr Ser Asn ValGlu Asp Asp Ser Thr Asn Gly Phe 260 265 270 His Asp Val Cys Gly Pro AsnLys Glu Leu Asp Glu Asp Thr Cys Gln 275 280 285 Cys Val Cys Lys Gly GlyLeu Arg Pro Ser Ser Cys Gly Pro His Lys 290 295 300 Glu Leu Asp Arg AspSer Cys Gln Cys Val Cys Lys Asn Lys Leu Phe 305 310 315 320 Pro Asn SerCys Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr Cys Gln 325 330 335 Cys ValCys Lys Arg Thr Cys Pro Arg Asn Gln Pro Leu Asn Pro Gly 340 345 350 LysCys Ala Cys Glu Cys Thr Glu Asn Thr Gln Lys Cys Phe Leu Lys 355 360 365Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr Arg Arg Pro Cys 370 375380 Ala Asn Arg Leu Lys His Cys Asp Pro Gly Leu Ser Phe Ser Glu Glu 385390 395 400 Val Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg Pro His Leu Asn405 410 415 22 base pairs nucleic acid single linear cDNA 42 TCTCTTCTGTGCTTGAGTTG AG 22 22 base pairs nucleic acid single linear cDNA 43TCTCTTCTGT CCCTGAGTTG AG 22 65 base pairs nucleic acid single linearcDNA 44 TGTGCTGCAG CAAATTTTAT AGTCTCTTCT GTGGCGGCGG CGGCGGCGGGCGCCTCGCGA 60 GGACC 65 30 base pairs nucleic acid single linear cDNA 45CTGGCAGGGA ACTGCTAATA ATGGAATGAA 30 84 base pairs nucleic acid singlelinear cDNA 46 GGGCTCCGCG TCCGAGAGGT CGAGTCCGGA CTCGTGATGG TGATGGTGATGGGCGGCGGC 60 GGCGGCGGGC GCCTCGCGAG GACC 84 31 base pairs nucleic acidsingle linear cDNA 47 GTATTATAAT GTCCTCCACC AAATTTTATA G 31 93 basepairs nucleic acid single linear cDNA 48 GTTCGCTGCC TGACACTGTGGTAGTGTTGC TGGCGGCCGC TAGTGATGGT GATGGTGATG 60 AATAATGGAA TGAACTTGTCTGTAAACATC CAG 93 18 base pairs nucleic acid single linear cDNA 49CATGTACGAA CCGCCAGG 18 20 base pairs nucleic acid single linear cDNA 50AATGACCAGA GAGAGGCGAG 20 10 amino acids amino acid single linear peptide51 Ala Val Val Met Thr Gln Thr Pro Ala Ser 1 5 10 196 amino acids aminoacid Not Relevant linear peptide 52 Met Arg Thr Leu Ala Cys Leu Leu LeuLeu Gly Cys Gly Tyr Leu Ala 1 5 10 15 His Val Leu Ala Glu Glu Ala GluIle Pro Arg Glu Val Ile Glu Arg 20 25 30 Leu Ala Arg Ser Gln Ile His SerIle Arg Asp Leu Gln Arg Leu Leu 35 40 45 Glu Ile Asp Ser Val Gly Ser GluAsp Ser Leu Asp Thr Ser Leu Arg 50 55 60 Ala His Gly Val His Ala Thr LysHis Val Pro Glu Lys Arg Pro Leu 65 70 75 80 Pro Ile Arg Arg Lys Arg SerIle Glu Glu Ala Val Pro Ala Val Cys 85 90 95 Lys Thr Arg Thr Val Ile TyrGlu Ile Pro Arg Ser Gln Val Asp Pro 100 105 110 Thr Ser Ala Asn Phe LeuIle Trp Pro Pro Cys Val Glu Val Lys Arg 115 120 125 Cys Thr Gly Cys CysAsn Thr Ser Ser Val Lys Cys Gln Pro Ser Arg 130 135 140 Val His His ArgSer Val Lys Val Ala Lys Val Glu Tyr Val Arg Lys 145 150 155 160 Lys ProLys Leu Lys Glu Val Gln Val Arg Leu Glu Glu His Leu Glu 165 170 175 CysAla Cys Ala Thr Thr Ser Leu Asn Pro Asp Tyr Arg Glu Glu Asp 180 185 190Thr Asp Val Arg 195 241 amino acids amino acid Not Relevant linearpeptide 53 Met Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu Cys Cys Tyr LeuArg 1 5 10 15 Leu Val Ser Ala Glu Gly Asp Pro Ile Pro Glu Glu Leu TyrGlu Met 20 25 30 Leu Ser Asp His Ser Ile Arg Ser Phe Asp Asp Leu Gln ArgLeu Leu 35 40 45 His Gly Asp Pro Gly Glu Glu Asp Gly Ala Glu Leu Asp LeuAsn Met 50 55 60 Thr Arg Ser His Ser Gly Gly Glu Leu Glu Ser Leu Ala ArgGly Arg 65 70 75 80 Arg Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro Ala MetIle Ala Glu 85 90 95 Cys Lys Thr Arg Thr Glu Val Phe Glu Ile Ser Arg ArgLeu Ile Asp 100 105 110 Arg Thr Asn Ala Asn Phe Leu Val Trp Pro Pro CysVal Glu Val Gln 115 120 125 Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn ValGln Cys Arg Pro Thr 130 135 140 Gln Val Gln Leu Arg Pro Val Gln Val ArgLys Ile Glu Ile Val Arg 145 150 155 160 Lys Lys Pro Ile Phe Lys Lys AlaThr Val Thr Leu Glu Asp His Leu 165 170 175 Ala Cys Lys Cys Glu Thr ValAla Ala Ala Arg Pro Val Thr Arg Ser 180 185 190 Pro Gly Gly Ser Gln GluGln Arg Ala Lys Thr Pro Gln Thr Arg Val 195 200 205 Thr Ile Arg Thr ValArg Val Arg Arg Pro Pro Lys Gly Lys His Arg 210 215 220 Lys Phe Lys HisThr His Asp Lys Thr Ala Leu Lys Glu Thr Leu Gly 225 230 235 240 Ala 149amino acids amino acid Not Relevant linear peptide 54 Met Pro Val MetArg Leu Phe Pro Cys Phe Leu Gln Leu Leu Ala Gly 1 5 10 15 Leu Ala LeuPro Ala Val Pro Pro Gln Gln Trp Ala Leu Ser Ala Gly 20 25 30 Asn Gly SerSer Glu Val Glu Val Val Pro Phe Gln Glu Val Trp Gly 35 40 45 Arg Ser TyrCys Arg Ala Leu Glu Arg Leu Val Asp Val Val Ser Glu 50 55 60 Tyr Pro SerGlu Val Glu His Met Phe Ser Pro Ser Cys Val Ser Leu 65 70 75 80 Leu ArgCys Thr Gly Cys Cys Gly Asp Glu Asn Leu His Cys Val Pro 85 90 95 Val GluThr Ala Asn Val Thr Met Gln Leu Leu Lys Ile Arg Ser Gly 100 105 110 AspArg Pro Ser Tyr Val Glu Leu Thr Phe Ser Gln His Val Arg Cys 115 120 125Glu Cys Arg Pro Leu Arg Glu Lys Met Lys Pro Glu Arg Cys Gly Asp 130 135140 Ala Val Pro Arg Arg 145 191 amino acids amino acid Not Relevantlinear peptide 55 Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu AlaLeu Leu Leu 1 5 10 15 Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala ProMet Ala Glu Gly 20 25 30 Gly Gly Gln Asn His His Glu Val Val Lys Phe MetAsp Val Tyr Gln 35 40 45 Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val AspIle Phe Gln Glu 50 55 60 Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro SerCys Val Pro Leu 65 70 75 80 Met Arg Cys Gly Gly Cys Cys Asn Asp Glu GlyLeu Glu Cys Val Pro 85 90 95 Thr Glu Glu Ser Asn Ile Thr Met Gln Ile MetArg Ile Lys Pro His 100 105 110 Gln Gly Gln His Ile Gly Glu Met Ser PheLeu Gln His Asn Lys Cys 115 120 125 Glu Cys Arg Pro Lys Lys Asp Arg AlaArg Gln Glu Asn Pro Cys Gly 130 135 140 Pro Cys Ser Glu Arg Arg Lys HisLeu Phe Val Gln Asp Pro Gln Thr 145 150 155 160 Cys Lys Cys Ser Cys LysAsn Thr Asp Ser Arg Cys Lys Ala Arg Gln 165 170 175 Leu Glu Leu Asn GluArg Thr Cys Arg Cys Asp Lys Pro Arg Arg 180 185 190 188 amino acidsamino acid Not Relevant linear peptide 56 Met Ser Pro Leu Leu Arg ArgLeu Leu Leu Ala Ala Leu Leu Gln Leu 1 5 10 15 Ala Pro Ala Gln Ala ProVal Ser Gln Pro Asp Ala Pro Gly His Gln 20 25 30 Arg Lys Val Val Ser TrpIle Asp Val Tyr Thr Arg Ala Thr Cys Gln 35 40 45 Pro Arg Glu Val Val ValPro Leu Thr Val Glu Leu Met Gly Thr Val 50 55 60 Ala Lys Gln Leu Val ProSer Cys Val Thr Val Gln Arg Cys Gly Gly 65 70 75 80 Cys Cys Pro Asp AspGly Leu Glu Cys Val Pro Thr Gly Gln His Gln 85 90 95 Val Arg Met Gln IleLeu Met Ile Arg Tyr Pro Ser Ser Gln Leu Gly 100 105 110 Glu Met Ser LeuGlu Glu His Ser Gln Cys Glu Cys Arg Pro Lys Lys 115 120 125 Lys Asp SerAla Val Lys Pro Asp Ser Pro Arg Pro Leu Cys Pro Arg 130 135 140 Cys ThrGln His His Gln Arg Pro Asp Pro Arg Thr Cys Arg Cys Arg 145 150 155 160Cys Arg Arg Arg Ser Phe Leu Arg Cys Gln Gly Arg Gly Leu Glu Leu 165 170175 Asn Pro Asp Thr Cys Arg Cys Arg Lys Leu Arg Arg 180 185 25 basepairs nucleic acid single linear cDNA 57 CCCGAGGATC GAGAATTAAT TCCCC 2523 base pairs nucleic acid single linear cDNA 58 AAAAAGCGGC CGATCCTCTAGAG 23

What is claimed is:
 1. A purified and isolated polypeptide that binds tothe extracellular domain of human Flt4 receptor tyrosine kinase(Flt4EC), said polypeptide including a contiguous portion of SEQ ID NO:33 that is sufficient to bind human Flt4EC, wherein said contiguousportion includes eight cysteine residues that are conserved in humanvascular endothelial growth factor (VEGF), human platelet derived growthfactor A (PDGF-A), and human platelet derived growth factor B (PDGF-B),and wherein said polypeptide lacks any portion of SEQ ID NO: 33 that hasone or more cysteine motifs of a Balbiani ring 3 protein (BR3P).
 2. Apolypeptide according to claim 1 further comprising a detectable label.3. A composition comprising a polypeptide according to claim 1 in apharmaceutically-acceptable diluent, adjuvant, excipient or carrier. 4.A purified and isolated mammalian polypeptide that binds to theextracellular domain of human Flt4 receptor tyrosine kinase (Flt4EC),said polypeptide including a contiguous portion of a mammalian Flt4ligand precursor polypeptide that is sufficient to bind human Flt4EC,wherein said mammalian Flt4 ligand precursor polypeptide is encoded by aDNA which hybridizes to a non-coding strand complementary to nucleotides352 to 1611 of SEQ ID NO: 32 under the following hybridizationconditions: hybridization at 42° C. in a hybridization solutioncomprising 50% formamide, 5×SSC, 20 mM Na.PO₄, pH 6.8; and washing in0.2×SSC at 55° C., wherein said contiguous portion includes eightcysteine residues that are conserved in human vascular endothelialgrowth factor (VEGF), human platelet derived growth factors A and B(PDGF-A, PDGF-B), human placenta growth factor (PlGF-1), and humanvascular endothelial growth factor B (VEGF-B), and wherein saidpolypeptide lacks any portion of the mammalian Flt4 ligand precursorpolypeptide that has one or more cysteine motifs of a Balbiani ring 3protein (BR3P).
 5. A purified and isolated polypeptide according toclaim 4, said polypeptide having a molecular weight of approximately 32kD as determined by SDS-PAGE under reducing conditions.
 6. A purifiedand isolated polypeptide according to claim 5, wherein said polypeptidestimulates tyrosine phosphorylation of human Flt4 receptor tyrosinekinase in a host cell expressing said Flt4 receptor tyrosine kinase. 7.A purified and isolated polypeptide according to claim 5, saidpolypeptide comprising an amino acid sequence set forth in SEQ ID NO:13.
 8. A purified and isolated polypeptide that binds to theextracellular domain of human Flt4 receptor tyrosine kinase, saidpolypeptide being encoded by plasmid pFLT4-L, deposited as ATCCAccession Number 97231, said polypeptide lacking the carboxyl terminalportion of the amino acid sequence encoded by plasmid pFLT4-L thatcontains cysteine motifs of a Balbiani ring 3 protein (BR3P).
 9. Apurified and isolated polypeptide that binds to the extracellular domainof human Flt4 receptor tyrosine kinase, said polypeptide having an aminoacid sequence consisting of a portion of the amino acid sequence setforth in SEQ ID NO: 33, said portion including from residue 161 of SEQID NO: 33 to residue 211 of SEQ ID NO: 33, said portion lacking at leastcarboxy-terminal residues of SEQ ID NO: 33 beyond residue
 227. 10. Apurified and isolated polypeptide according to claim 9 wherein saidportion of the amino acid sequence set forth in SEQ ID NO: 33 includesfrom residue 131 of SEQ D NO: 33 to residue 211 of SEQ ID NO:
 33. 11. Apurified and isolated polypeptide according to claim 9 wherein saidportion of the amino acid sequence set forth in SEQ ID NO: 33 includesfrom residue 113 of SEQ ID NO: 33 to residue 213 of SEQ ID NO:
 33. 12. Apurified and isolated polypeptide according to claim 9 wherein saidportion of the amino acid sequence set forth in SEQ ID NO: 33 includesamino acids 103 to 217 of SEQ ID NO:
 33. 13. A purified and isolatedpolypeptide according to claim 9 wherein said portion of the amino acidsequence set forth in SEQ ID NO: 33 includes amino acids 103 to 225 ofSEQ ID NO:
 33. 14. A purified and isolated polypeptide according toclaim 9 wherein said portion of the amino acid sequence set forth in SEQID NO: 33 includes amino acids 32 to 227 of SEQ ID NO:
 33. 15. Apurified and isolated polypeptide that binds to the extracellular domainof human Flt4 receptor tyrosine kinase, said polypeptide comprising aportion of the amino acid sequence in SEQ ID NO: 33 effective to permitsaid binding, said polypeptide lacking at least carboxy-terminalresidues of SEQ ID NO: 33 beyond residue
 227. 16. A polypeptideaccording to claim 15 wherein said portion of the amino acid sequence inSEQ ID NO: 33 is a continuous portion having as its amino terminalresidue an amino acid between residues 30 and 162 of SEQ ID NO: 33 andhaving as its carboxy terminal residue an amino acid between residues210 and 228 of SEQ ID NO:
 33. 17. A polypeptide according to claim 15wherein said portion of the amino acid sequence in SEQ ID NO: 33 is acontinuous portion having as its amino terminal residue an amino acidbetween residues 102 and 132 of SEQ ID NO: 33 and having as its carboxyterminal residue an amino acid between residues 210 and 228 of SEQ IDNO:
 33. 18. A polypeptide according to claim 15 wherein said portion ofthe amino acid sequence in SEQ ID NO: 33 is a continuous portion having,as its amino terminal residue, residue 103 of SEQ ID NO: 33; and having,as its carboxy terminal residue, an amino acid between residues 216 and228 of SEQ ID NO:
 33. 19. A purified protein comprising a firstpolypeptide linked to a second polypeptide, wherein at least one of saidfirst polypeptide and said second polypeptide is a polypeptide accordingto claim 15, 16, 17, or 18, and wherein said purified protein binds tothe extracellular domain of Flt4 receptor tyrosine kinase.
 20. Apurified protein comprising a first polypeptide linked to a secondpolypeptide, wherein each of said first polypeptide and said secondpolypeptide is a polypeptide according to claim 15, 16, 17, or 18, andwherein said purified protein binds to the extracellular domain of Flt4receptor tyrosine kinase.
 21. A polypeptide according to any one ofclaims 15-18, wherein said polypeptide further includes a polyhistidineamino acid sequence.
 22. A polypeptide according to claim 15 having anapparent molecular weight of approximately 32 kD as assessed by SDS-PAGEunder reducing conditions.
 23. A polypeptide according to claim 15 thatbinds the extracellular domain of Flt4 receptor tyrosine kinase andstimules Flt4 phosphorylation in mammalian cells expressing Flt4receptor tyrosine kinase.
 24. A purified and isolated polypeptideaccording to claim 15 wherein said portion of the amino acid sequence inSEQ ID NO: 33 is a continuous portion having, as its amino terminalresidue, residue 103 of SEQ ID NO: 33; and having, as its carboxylterminal residue, an amino acid between residues 221 and 228 of SEQ IDNO:
 33. 25. A purified polypeptide according to claim 15, wherein theamino acid sequence of said polypeptide consists of a portion of theamino acid sequence in SEQ ID NO:
 33. 26. A purified polypeptideaccording to claim 25 wherein said polypeptide has an apparent molecularweight of approximately 21-23 kD as assessed by SDS-PAGE under reducingconditions.
 27. A purified polypeptide according to claim 25 whereinsaid polypeptide has an apparent molecular weight of about 32 kD asassessed by SDS-PAGE under reducing conditions.
 28. A purified proteincomprising a first polypeptide linked to a second polypeptide, whereinat least one of said first polypeptide and said second polypeptide is apolypeptide according to claim 25, and wherein said protein is capableof binding to the extracellular domain of human Flt4 receptor tyrosinekinase.
 29. A purified protein according to claim 28 wherein said firstpolypeptide is covalently linked to said second polypeptide.
 30. Apurified protein according to claim 28 wherein the amino acid sequenceof at least one of said first polypeptide and said second polypeptide isan amino acid sequence selected from the group consisting of: (a) aminoacids 161 to 211 of SEQ ID NO: 33; (b) amino acids 131 to 211 of SEQ IDNO: 33; (c) amino acids 113 to 213 of SEQ ID NO: 33; (d) amino acids 113to 227 of SEQ ID NO: 33; (e) amino acids 103 to 217 of SEQ ID NO: 33;(f) amino acids 103 to 225 of SEQ ID NO: 33; (g) amino acids 103 to 227of SEQ ID NO: 33; and (h) amino acids 32 to 227 of SEQ ID NO:
 33. 31. Apurified protein according to claim 28 wherein the amino acid sequencesof said fist polypeptide and said second polypeptide are selected fromthe group consisting of: (a) amino acids 161 to 211 of SEQ ID NO: 33;(b) amino acids 131 to 211 of SEQ ID NO: 33; (c) amino acids 113 to 213of SEQ ID NO: 33; (d) amino acids 113 to 227 of SEQ ID NO: 33; (e) aminoacids 103 to 217 of SEQ ID NO: 33, (e) amino acids 103 to 225 of SEQ IDNO: 33; (g) amino acids 103 to 227 of SEQ ID NO: 33; (g) amino acids 32to 227 of SEQ ID NO: 33; and (i) amino acids 228 to 419 of SEQ ID NO:33.
 32. A purified and isolated polypeptide that binds to theextracellular domain of human Flt4 receptor tyrosine kinase, saidpolypeptide comprising a portion of SEQ ID NO: 33 effective to permitsaid binding, said portion of SEQ ID NO: 33 lacking at least carboxyterminal residues of SEQ ID NO: 33 having cysteine motifs of a Balbianiring 3 protein.
 33. A purified polypeptide that binds to theextracellular domain of human Flt4 receptor tyrosine kinase, saidpolypeptide produced by a method comprising the steps of: (a) expressinga nucleic acid in a host cell; and (b) purifying a polypeptide thatbinds to the extracellular domain of human Flt4 receptor tyrosine kinasefrom said host cell or from a growth medium of said host cell; whereinsaid nucleic acid comprises a nucleotide sequence encoding a portion ofthe amino acid sequence in SEQ ID NO: 33 effective to permit suchbinding, and wherein said polypeptide lacks at least thecarboxy-terminal portion of SEQ ID NO: 33 that is characterized bycysteine motifs of a Balbiani ring 3 protein.
 34. A purified andisolated polypeptide according to claim 33, wherein said purifyingcomprises an affinity purification procedure wherein the affinitypurification matrix comprises a polypeptide comprising the extracellulardomain of Flt4 receptor tyrosine kinase.
 35. A polypeptide according toclaim 33, that has an apparent molecular weight of approximately 32 kDas assessed by SES-PAGE under reducing conditions.
 36. A polypeptideaccording to claim 33 wherein said portion of the amino acid sequence inSEQ ID NO: 33 encoded by said polynucleotide is a continuous portionhaving as its amino terminal residue an amino acid between residues 30and 162 of SEQ ID NO: 33 and having as its carboxy terminal residue anamino acid between residues 210 and 228 of SEQ ID NO:
 33. 37. Apolypeptide according to claim 33 wherein said portion of the amino acidsequence in SEQ ID NO: 33 encoded by said polynucleotide is a continuousportion having as its amino terminal residue an amino acid betweenresidues 102 and 132 of SEQ ID NO: 33 and having as its carboxy terminalresidue an amino acid between residues 210 and 228 of SEQ ID NO:
 33. 38.A polypeptide according to claim 33 wherein said portion of the aminoacid sequence in SEQ ID NO: 33 encoded by said polynucleotide is acontinuous portion having, as its amino terminal residue, residue 103 ofSEQ ID NO: 33, and having, as its carboxy terminal residue, an aminoacid between residues 216 and 228 of SEQ ID NO:
 33. 39. A purified andisolated polypeptide having the amino acid sequence of residues 1 to 415of SEQ ID NO:
 41. 40. A purified and isolated polypeptide that binds tothe extracellular domain of an Flt4 receptor tyrosine kinase, saidpolypeptide comprising a contiguous portion of the amino acid sequenceof SEQ ID NO: 41 effective to permit such binding, said contiguousportion including eight cysteine residues of SEQ ID NO: 41 that areconserved in human vascular endothelial growth factor (VEGF), humanplatelet derived growth factors A and B (PDGF-A, PDGF-B), human placentagrowth factor (PlGF-1), and human vascular endothelial growth factor B(VEGF-B).
 41. A purified polypeptide that binds to the extracellulardomain of human Flt4 receptor tyrosine kinase, said polypeptide producedby a method comprising the steps of: (a) expressing a nucleic acid in ahost cell, said nucleic acid comprising a nucleotide sequence encoding aportion of the amino acid sequence in SEQ ID NO: 41 effective to permitsuch binding; and (b) purifying a polypeptide that binds to theextracellular domain of human Flt4 receptor tyrosine kinase from saidhost cell or from a growth medium of said host cell, wherein saidpolypeptide lacks at least the carboxy-terminal portion of SEQ ID NO: 41that is characterized by cysteine motifs of a Balbiani ring 3 protein.42. A composition comprising a polypeptide according to any one ofclaims 4, 8-11, 15-18, 24, 32, 33, and 36-37 in apharmaceutically-acceptable diluent, adjuvant, excipient, or carrier.43. A method of modulating the activity of human Flt4 receptor tyrosinekinase comprising administering to a person in need of modulation ofFlt4 receptor tyrosine kinase activity a composition according to claim42.
 44. A method of modulating the activity of human Flt4 receptortyrosine kinase comprising contacting cells that express human Flt4receptor tyrosine kinase with a polypeptide according to any one ofclaims 4, 8-11, 15-18, 24, 32, 33 and 36-37.
 45. A method of modulatingthe growth of mammalian endothelial cells comprising the steps of:exposing mammalian endothelial cells to a polypeptide according to anyone of claims 4, 8-11, 15-18, 24, 32, 33 and 36-37, in an amounteffective to modulate the growth of mammalian endothelial cells, whereinthe mammalian endothelial cells comprise cells that express Flt4receptor tyrosine kinase.
 46. A method for detecting endothelial cellsthat express Flt4 in a biological tissue comprising the steps ofexposing a biological tissue comprising endothelial cells to apolypeptide according to any one of claims 4, 8-11, 15-18, 24, 32, 33and 36-37, under conditions wherein said polypeptide binds toendothelial cells; and detecting said polypeptide bound to endothelialcells in said biological tissue, thereby detecting said endothelialcells.